Team:BIOSINT Mexico/lab records

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   <h1 style="margin-top:20px;margin-bottom:5px">Protocols</h1>
+
   <h1 style="margin-top:20px;margin-bottom:5px">Lab Records</h1>
   <div class="leftside">
   <div class="leftside">
     <ul>
     <ul>
-
       <li><a onclick="showprot('#prot1')">Preparation of Terrific Broth and LB Agar</a></li>
+
       <li><a onclick="showprot('#prot1')"> JUNE</a></li>
-
       <li><a onclick="showprot('#prot2')">Preparation of MS media for Arabidopsis thaliana</a></li>
+
       <li><a onclick="showprot('#prot2')"> JULY </a></li>
-
       <li><a onclick="showprot('#prot3')">Preparation of Antibiotic stocks</a></li>
+
       <li><a onclick="showprot('#prot3')"> AUGUST </a></li>
-
       <li><a onclick="showprot('#prot4')">CaCl2 Competent Cells</a></li>
+
       <li><a onclick="showprot('#prot4')"> SEPTEMBER </a></li>
-
       <li><a onclick="showprot('#prot5')">Heat Shock Transformation of E. coli</a></li>
+
       <li><a onclick="showprot('#prot5')"> OCTOBER </a></li>
-
       <li><a onclick="showprot('#prot6')">Transformation by Electroporation</a></li>
+
        
-
      <li><a onclick="showprot('#prot7')">Plasmid purification by Miniprep</a></li>
+
-
      <li><a onclick="showprot('#prot8')">Plasmid purification by Midiprep</a></li>
+
-
      <li><a onclick="showprot('#prot9')">Electrophoresis Agarose Gel</a></li>
+
-
      <li><a onclick="showprot('#prot10')">Restriction Digest</a></li>
+
-
      <li><a onclick="showprot('#prot11')">Parts Ligation</a></li>
+
-
      <li><a onclick="showprot('#prot12')">Gel DNA Purification</a></li>
+
-
      <li><a onclick="showprot('#prot15')">REFERENCIAS </a></li>
+
     </ul>
     </ul>
   </div>
   </div>
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   <div class="rightside">
   <div class="rightside">
     <div id="prot1" class="protocolactive">
     <div id="prot1" class="protocolactive">
-
       <h2> Preparation of <i>Terrific Broth and LB Agar</i></h2>
+
       <h3> 3 June</h3>
-
       <p><ol>
+
<h4>Team meeting</h4>
-
  <li>Weight 35 grams of LB Agar (Sigma-Aldrich) for each liter of media.</li>
+
<br> Notes: Summer lab Schedule!
-
  <li>Weight 42.5 grams of Terrific Broth (Sigma-Aldrich) for each liter of media.</li>
+
       <h3> 4 June</h3>
-
  <li>Dilute in Erlenmeyer flasks with the adequate amount of distilled water.</li>
+
<h4>Media preparation LB broth and LB agar and E.coli inoculation</h4>
-
  <li>Heat in constant agitation.</li>
+
<b>Procedure:</b>
-
  <li>Place the flasks in the autoclave with autoclave tape.</li>
+
<br>* 14g of LB agar were weighted to prepare 400ml of medium
-
  <li>Set te autoclave until it reaches 120 °C and 15 PSI.</li>
+
<br>* 1 g of LB broth were weighted to prepare 500 ml of medium
-
  <li>Store both the Broth and Agar at 4 °C.</li>
+
<br>* Autoclave at 121°C for 15 minutes
-
        </ol></p>
+
<br>* E.coli was inoculated in petri dishes and incubated at 37°C
-
    </div>
+
<br><b>Notes:</b> The growth mediums in storage were revised; they didn’t show signs of contamination
 +
<h4>Calcium chloride for competent cells </h4>
 +
<b>Procedure:</b>
 +
<br>* Sterilize distilled water in the autoclave for 15 minutes at 121⁰c
 +
<br>* Dilute 1.1159 g of calcium chloride in 50 ml of sterile distilled water
 +
<br>* Fill a syringe to its maximum capacity and connect a ministart 0.2µm filter
 +
<br>* Pour the content of the syringe into a sterile falcon tube through the filter
 +
      <h3> 5 June</h3>
 +
<h4>E.coli Competent cells</h4>
 +
<b>Procedure:</b>
 +
<br>* Add 1.5 ml of E.coli in LB broth to Ice tubes
 +
<br>* Centrifuged for 3 minutes at 6000 rpm, two times
 +
<br>* The supernatant was discarded
 +
<br>* The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
 +
<br>* Incubated in ice for 20 minutes
 +
<br>* The tubes were centrifuged again for 3 minutes at 6000 rpm
 +
<br>* The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
 +
<br>* They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c
 +
<br>
 +
<h4>MS media preparation for Arabidopsis</h4>
 +
<b>Procedure:</b>
 +
<br>* For 500 ml of MS media measure:5 g of sucrose, 2.2 g of basal MS salt, 4 g of agar, 5 ml of Vitamin Gamblor and 500 ml of distilled water
 +
<br>* Mix all the components, except the agar, in constant agitation.
 +
<br>* Adjust the pH to 5.7 ± 0.1.
 +
<br>* Add the agar and mix gently and heat until it reaches 65 – 70 °C. (Do not boil or autoclave)
 +
<br>* Pour the agar in plates, let them cool and store at 4 °C.
-
    <div id="prot2" class="protocol">
+
<h3> 6 June </h3>
-
      <h2> Preparation of MS media for Arabidopsis thaliana </h2>
+
<h4>Transformation of E.coli with MerB and MerE (from DNA synthesis)</h4>
-
      <p>For 500 ml of MS (Murashige-Skoog) media measure: </p>
+
-
      <ul>
+
-
              <li>5 g of sucrose</li>
+
-
              <li>2.2 g of basal MS salt (Sigma-Aldrich)</li>
+
-
              <li>4 g of agar</li>
+
-
<li>5 ml of Vitamin stock mix (Gamblor stock solution)</li>
+
-
<li>500 ml of distilled water</li>
+
-
    </ul>
+
-
      <p><ol>
+
<b>Procedure:</b>
-
  <li>Mix all the components, except the agar, in constant agitation.</li>
+
<br>*50 µl of competent cells were placed in a cold eppendorf tube
-
  <li> Adjust the pH to 5.7 ± 0.1.</li>
+
<br>*4 µl of DNA sample were added
-
  <li> Add the agar and mix gently and heat until it reaches 65 – 70 °C. (Do not boil or autoclave)</li>
+
<br>*It was mixed gently and left incubating in ice for 30 minutes
-
  <li>Pour the agar in plates, let them cool and store at 4 °C.</li>
+
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
-
+
<br>*The tube was placed in ice for 2-5 minutes
-
      </ol></p>
+
<br>*900 µl of S.O.C. were added
-
    </div>
+
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and ampicillin(100mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
-
    <div id="prot3" class="protocol">
+
<b><br>Notes:</b>The concentration of lyophilized DNA from synthesis was 200ng, rehydrated with 1 ml of elution buffer, for a final concentration of 2ng/10 ul.  
-
      <h2>Preparation of Antibiotic stocks </h2>
+
E.coli inoculation from SOC media in LB dishes with ampicillin, will be done tomorrow
-
      <ul>
+
The first colonies appear three days later. The transformation efficiency of competent cells should be review.
-
              <li>Cloramphenicol Stock:</li>
+
-
            </ul>     
+
-
    <p><ol>
+
-
  <li>Weight 10 mg of lyophilized antibiotic for each ml of stock solution.</li>
+
-
          <li>Dissolve in ethanol and mix gently (It is possible to use Vortex)</li>
+
-
      </ol></p>
+
-
<ul>
+
      <h3> 9 June</h3>
-
              <li>Kanamicin Stock:</li>
+
<h4>Growing arabidopsis seeds in media</h4>
-
            </ul>    
+
<b>Procedure:</b>
-
    <p><ol>
+
<br><b>Notes: </b>
-
  <li>Weight 50 mg of lyophilized antibiotic for each ml of stock solution.</li>
+
<h4>Biobricks rehydration and transformation of E.coli: PhyB, Pif6, NLS, VP16 and RFP 10 pg( transformation efficiency)</h4>
-
          <li>Dissolve in distilled water and mix gently (It is possible to use Vortex).</li>
+
<b>Procedure:</b>
-
      </ol></p>
+
<br>* Kit plates were rehydrated with 10 ul of distillated water
 +
<br>* 50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>* 2 µl of DNA sample were added
 +
<br>* It was mixed gently and left incubating in ice for 30 minutes
 +
<br>* The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>* The tube was placed in ice for 2-5 minutes
 +
<br>* 900 µl of S.O.C. were added
 +
<br>* The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>* A dish with LB agar and Chloramphenicol (35mg/ml) was striated with the transformed cells
 +
<br>* The dish was sealed and left incubating at 37⁰c
 +
<br>
 +
<br><b>Notes:</b>The first colonies appear two days later and transformation efficiency kit is not working as expected. The transformation efficiency of competent cells should be review at different concentration.
 +
<br>Antibiotic concentration should be review.
 +
<br>
 +
 
 +
 
 +
<h3> 10 June</h3>
 +
<h4>Miniprep MerB and MerE (sample from DNA synthesis)</h4>
 +
<b>Procedure:</b>
 +
<br>* Centrifuge E,coli transformed culture at 12000 rpm for 1 minute two times
 +
<br>* Resuspend in 250 µl resuspension buffer
 +
<br>* Add 250 µl of Lysis Buffer L7
 +
<br>* Add 350 µl of precipitation Buffer N4
 +
<br>* Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>* Centrifuge at 12000 rpm for a minute
 +
<br>* The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>* The column was centrifuged at 12000 rpm for 1 minute
 +
<br>* 700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>* The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>* 75 µl of preheated TE Buffer were added
 +
<br>* The column was centrifuged at 12000 rpm for 2 minutes
 +
<br><b>Notes: </b>Inventory season!, running gels must wait until next week
 +
 
 +
<h3>11 june</h3>
 +
 
 +
<h4>Miniprep PhyB, Pif6, NLS, VP16 and RFP 10 pg (Transformation efficiency)</h4>
 +
 
 +
<b>Procedure:</b>
 +
<br> *Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br> *Resuspend in 250 µl resuspension buffer
 +
<br> *Add 250 µl of Lysis Buffer L7
 +
<br> *Add 350 µl of precipitation Buffer N4
 +
<br> *Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br> *Centrifuge at 12000 rpm for a minute
 +
<br> *The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br> *The column was centrifuged at 12000 rpm for 1 minute
 +
<br> *700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br> *The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br> *75 µl of preheated TE Buffer were added
 +
<br> *The column was centrifuged at 12000 rpm for 2 minutes
 +
 
 +
<b><br>Notes:</b>Inventory season!, running gels must wait until next week
 +
 
 +
<h3>13 june</h3>
 +
 
 +
<h4>Media preparation (LB broth and LB agar)</h4>
 +
<b>Procedure</b>
 +
<br> *14g of LB agar were weighted to prepare 400ml of medium
 +
<br> *1 g of LB broth were weighted to prepare 500 ml of medium
 +
<br> *Autoclave at 121°C for 15 minutes
 +
 
 +
<h3>17 june</h3>
 +
 
 +
<h4>Restriction digest of MerE, MerB and PSB1C3</h4>
 +
 
 +
<b>Procedure:</b>
 +
<br>*Add 10 ul of DNA to be digested, and 6 ul of dH20
 +
<br>*Add 2.5ul of NEBuffer 2.
 +
<br>*Add 0.5ul of BSA.
 +
<br>*Add 0.5ul of EcoRI.
 +
<br>*Add 0.5ul of PstI.
 +
<br>*Mix well and spin down briefly.
 +
<br>*Incubate the restriction digest at 37C for 30min, and then 80C for 20min
 +
 
 +
<b><br>Notes:</b>Mislabelling of sample, the experiment must be repeated
 +
 
 +
<h4>Ligation of MerE and  MerB in PSB1C3</h4>
 +
 
 +
<b>Procedure</b>
 +
<br>*Mix well and spin down briefly.
 +
<br>*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample, 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
 +
<br>*Incubate 1 hour at 22°C. Inactivation of T4 DNA ligase at 65°C for 10 min
 +
<br>*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
 +
 
 +
<b><br>Notes:</b> T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
 +
 
 +
<br>Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants
 +
 
 +
<br>Mislabelling of sample, the experiment must be repeated
 +
 
 +
<h3>18 june </h3>
 +
 
 +
<h4>Restriction digest of MerE, MerB and PSB1C3 </h4>
 +
 
 +
<b>Procedure:</b>
 +
<br>*Add 10 ul of DNA to be digested, and 6 ul of dH20
 +
<br>*Add 2.5ul of NEBuffer 2.
 +
<br>*Add 0.5ul of BSA.
 +
<br>*Add 0.5ul of EcoRI.
 +
<br>*Add 0.5ul of PstI.
 +
<br>*Mix well and spin down briefly.
 +
<br>*Incubate the restriction digest at 37C for 30min, and then 80C for 20min
 +
 
 +
<h4>Ligation of MerE and  MerB in PSB1C3</h4>
 +
<b>Procedure</b>
 +
 
 +
<br>*Mix well and spin down briefly.
 +
<br>*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample, 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
 +
<br>*Incubate 1 hour at 22°C.
 +
<br>*inactivation of T4 DNA ligase at 65°C for 10 min
 +
<br>*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
 +
 
 +
<b><br>Notes:</b>T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
 +
 
 +
<br>Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants
 +
 
 +
<h4>Gel electrophoresis: PhyB, MerE, VP16, NLS, PIf6, MerB, RFP and PSB1C3</h4>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/3/3d/Biogel1.JPG" widht="30" height="400"/>
 +
 
 +
 
 +
<b><br>Procedure:</b>
 +
<br>*2% agarose gel, the current was set to 100 V for 55 minutes.
 +
 
 +
<b>Results:</b>
 +
 
 +
<b><br>Notes:</b>No band appears, there is not DNA sample in the gel. The transformation experiments must be repeated.
 +
 
 +
<h3>19 june </h3>
 +
 
 +
<h4>Gel Electrophoresis: MerB and MerE Restriction digest and ligation </h4>
 +
 
 +
<b>Procedure:</b>
 +
 
 +
<br>*2% agarose gel, the current was set to 100 V for 55 minutes.
 +
 
 +
<b>Results:</b>
 +
<img src="https://static.igem.org/mediawiki/2014/8/88/Biogel2.JPG" widht="30" height="400"/>
 +
<b><br>Notes:</b>No band appears, there is not DNA sample in the gel. The transformation, restriction digests and ligation experiments must be repeated.
 +
 
 +
<h3>20 june</h3>
 +
 
 +
<h4>Transformation of E.coli with MerB and MerE in PSB1C3</h4>
 +
<b>Procedure</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>*The tube was placed in ice for 2-5 minutes
 +
<br>*900 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Ampicillin (100mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 
 +
<b><br>Notes:</b>To confirm that the gel has done correctly, parts ligated with PSB1C3 were transformed in E,coli
 +
After one week, no colonies growth in petri dishes
 +
 
 +
<h3>23 june </h3>
 +
 
 +
<h4>Competent cells</h4>
 +
 
 +
<b>Procedure</b>
 +
<br>*Add 1.5 ml of E.coli in LB broth to Ice tubes
 +
<br>*Centrifuged for 3 minutes at 6000 rpm, two times
 +
<br>*The supernatant was discarded
 +
<br>*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
 +
<br>*Incubated in ice for 20 minutes
 +
<br>*The tubes were centrifuged again for 3 minutes at 6000 rpm
 +
<br>*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
 +
<br>*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c
 +
<b><br>Note: </b>The first competent cells didn’t work as expected. The same protocol was applied this time.
 +
 
 +
<h4>Test 1: Transformation of E.coli with MerB, MerE, NLS, PhyB, VP16, AGS linker, PIF6, PolyA, TetR, RFP 10 pg (transformation efficiency)</h4>
 +
 
 +
<b>Procedure</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*2 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>*The tube was placed in ice for 2-5 minutes
 +
<br>*900 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Chloramphenicol (35mg/ml) or Ampicillin(100mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 
 +
<b><br>Notes: </b>The next day no colony appears. A second culture of the same SOC solution was done.  
 +
The first colonies of MerB, MerE from the first culture appears two days later and the next day the second culture grew. But transformation efficiency kit and parts from the distribution kit are not working as expected.
 +
<br>Some colonies of Pif6 and RFP grew, after three days of incubation.
 +
<br>Antibiotic concentration of Chloramphenicol and competent cells efficiency should be review.
 +
 
 +
<h4>Terrific Broth preparation </h4>
 +
 
 +
<b>Procedure:</b>
 +
 
 +
<br>*Weight 42.5 grams of Terrific Broth (Sigma-Aldrich) for each liter of media.
 +
<br>*Dilute in Erlenmeyer flasks with the adequate amount of distilled water.
 +
<br>*Autoclave
 +
 
 +
<h4>Pasar arabidopsis a tierra y medio </h4>
 +
 
 +
 
 +
<h3>24 june</h3>
 +
 
 +
<h4>Inoculation of Transformed colonies of Test 1 in terrific broth (MerB, MerE, Pif6 and RFP)</h4>
 +
 
 +
<b>Procedure:</b>
 +
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
 +
 
 +
<h4>Media preparation: LB agar with antibiotics at different concentrations  </h4>
 +
 
 +
<h3>25 june</h3>
 +
 
 +
<h4>Competent cells</h4>
 +
 
 +
<b>Procedure: </b>
 +
<br>*Add 1.5 ml of E.coli in LB broth to Ice tubes
 +
<br>*Centrifuged for 3 minutes at 6000 rpm, two times
 +
<br>*The supernatant was discarded
 +
<br>*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
 +
<br>*Incubated in ice for 20 minutes
 +
<br>*The tubes were centrifuged again for 3 minutes at 6000 rpm
 +
<br>*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
 +
<br>*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c
 +
<b><br>Notes: </b> After the problems with test1, efficiency of competent cells it was not as expected. New competent cells was made.
 +
 
 +
<h4>Miniprep (1) MerB, MerE, RFP(te),Pif6 </h4>
 +
 
 +
<b>Aim of the experiment: </b>
 +
 
 +
<br>*Plasmid extraction from colonies of “transformation (test 1)”
 +
 
 +
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
 +
 
 +
<b><br>Notes:</b>
 +
 
 +
 
 +
 
 +
<h4>Test 2: Transformation of E.coli with MerB, MerE, NLS, PhyB, VP16, AGS linker, PIF6, PolyA, TetR, RFP(control)</h4>
 +
 
 +
<b>Procedure:</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*2 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>*The tube was placed in ice for 2-5 minutes
 +
<br>*900 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and  Chloramphenicol (15mg/ml) / Ampicillin (100 mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 
 +
<b><br>Notes:</b>The colonies of MerB, MerE, NLS, AGS, Pif6, VP16 and TetR from the first culture appear the next day. But transformation efficiency kit and parts from the distribution kit are not working as expected.
 +
Antibiotic concentration of Chloramphenicol was changed to (15 mg/ml).
 +
 
 +
<h3>26 june</h3>
 +
 
 +
<h4>Miniprep (2) MerB, MerE, NLS, AGS, Pif6, VP16, TetR</h4>
 +
 
 +
<b>Aim of the experiment: </b>
 +
 
 +
<h4>Plasmid extraction from colonies of “transformation (test 2)”</h4>
 +
 
 +
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
 +
 
 +
<h3>27 june </h3>
 +
 
 +
<h4>Electrophoresis Gel of MerE, MerB, RFP</h4>
 +
<b><br>Results:</b>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/b/b2/Biogel3.JPG" widht="30" height="400"/>
 +
 
 +
<h3>30 june </h3>
 +
 
 +
<h4>Electrophoresis gel of Miniprep: NLS, AGS, Pif6, VP16, TetR</h4>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/d/d9/Biogel4.JPG" widht="30" height="400"/>
 +
 
 +
</div>
 +
 
 +
 
 +
<div id="prot2" class="protocolactive">
 +
 
 +
<h3>1 july </h3>
 +
 
 +
<h4>Test1: Restriction digest TetR, Pif6, MerB, NLS, PolyA, MerE, MerB, PSB1C3</h4>
 +
 
 +
<b>Procedure</b>
 +
<br>***Note: some parts are A/B, cut them with EcoRI and PstI
 +
<br>*Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes
 +
<br>*Add 10 ul of DNA to be digested, and 6 ul of dH20
 +
<br>*Pipet 2.5ul of NEB Buffer 2 to each tube.
 +
<br>*Pipet 0.5ul of BSA to each tube.
 +
<br>*In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.
 +
<br>*In the Part B tube: Add 0.5ul of XbaI, and 0.5ul of PstI.
 +
<br>*In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
 +
<br>*Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture
 +
<br>*Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.
 +
 
 +
<h3>2 july</h3>
 +
 
 +
<h4>Test 1: Ligation </h4>
 +
 
 +
<b>Procedure</b>
 +
<br>*Mix well and spin down briefly.
 +
<br>*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample  (4ul for sample A, 4 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
 +
<br>*Incubate 1 hour at 22°C.
 +
<br>*inactivation of T4 DNA ligase at 65°C for 10 min
 +
<br>*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
 +
 
 +
<b><br>Notes: </b>T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
 +
 
 +
<br>Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants
 +
 
 +
<br>Electrophoresis Gel  (same samples as 30 july) and more samples
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/4/4b/Biogel5.JPG" widht="30" height="400"/>
 +
 
 +
<h3>3 july </h3>
 +
 
 +
<h4>Electrophoresis gel: Restriction digests gel of Test 1</h4>
 +
<img src="https://static.igem.org/mediawiki/2014/a/a6/Biogel6.JPG" widht="30" height="400"/>
 +
 
 +
<h4>Electrophoresis gel: Ligation of test 1</h4>
 +
<img src="https://static.igem.org/mediawiki/2014/c/c2/Biogel7.JPG" widht="30" height="400"/>
 +
 
 +
<h3>4 july </h3>
 +
 +
<h4>Test 2: Restriction digest </h4>
 +
 
 +
<br>***Note: for parts are A/B, cut with EcoRI and PstI
 +
<br>*Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes
 +
<br>*Add 10 ul of DNA to be digested, and 6 ul of dH20
 +
<br>*Pipet 2.5ul of NEB Buffer 2 to each tube.
 +
<br>*Pipet 0.5ul of BSA to each tube.
 +
<br>*In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.
 +
<br>*In the Part B tube: Add 0.5ul of XbaI, and 0.5ul of PstI.
 +
<br>*In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
 +
<br>*Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture
 +
<br>*Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.
 +
 
 +
<h3>7 july </h3>
 +
 
 +
<h4>Test 2: Ligation</h4>
 +
 
 +
<b>Procedure</b>
 +
<br>*Mix well and spin down briefly.
 +
<br>*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample  (4ul for sample A, 4 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
 +
<br>*Incubate 1 hour at 22°C.
 +
<br>*inactivation of T4 DNA ligase at 65°C for 10 min
 +
<br>*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
 +
 
 +
<b><br>Notes: </b>T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
 +
 
 +
<br>Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants
 +
 
 +
<h3>8 july </h3>
 +
 
 +
<h4>Electrophoresis gel: Restriction digest of test 2</h4>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/2/21/Biogel8.JPG" widht="30" height="400"/>
 +
 
 +
<h4>Electrophoresis gel: Ligation of Test 2 </h4>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/9/9f/Biogel9.JPG" widht="30" height="400"/>
 +
 
 +
<h3>9 july </h3>
 +
 
 +
<h4>Transformation of E.coli with Test 2</h4>
 +
 
 +
<b>Procedure</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*3 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>*The tube was placed in ice for 2-5 minutes
 +
<br>*900 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
<b><br>Notes:</b>
 +
 
 +
 
 +
<h4>Rehydratation and Transformation PhyB</h4>
 +
<br>*Kit plates were rehydrated with 10 ul of distillated water
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*2 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>*The tube was placed in ice for 2-5 minutes
 +
<br>*900 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 
 +
 +
<h3>10 july</h3>
 +
 
 +
<h4>Test for antibiotic concentration</h4>
 +
 
 +
<h3>14 july </h3>
 +
 
 +
<h4>Restriction digests of linear plasmid PSB1A3</h4>
 +
 
 +
<b>Procedure</b>
 +
<br>*Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tube
 +
<br>*Add 10 ul of DNA to be digested, and 6 ul of dH20
 +
<br>*Pipet 2.5ul of NEB Buffer 2
 +
<br>*Pipet 0.5ul of BSA
 +
<br>*Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
 +
<br>*Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture
 +
<br>*Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.
 +
 
 +
<h4>Electrophoresis gel of PhyB </h4>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/e/e4/Biogel10.JPGDiapositiva10.JPG" widht="30" height="400"/>
 +
 
 +
<h3>15 july </h3>
 +
 
 +
<h4>Competent cells </h4>
 +
 
 +
<b>Procedure:</b>
 +
<br>*Add 1.5 ml of E.coli in LB broth to Ice tubes
 +
<br>*Centrifuged for 3 minutes at 6000 rpm, two times
 +
<br>*The supernatant was discarded
 +
<br>*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
 +
<br>*Incubated in ice for 20 minutes
 +
<br>*The tubes were centrifuged again for 3 minutes at 6000 rpm
 +
<br>*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
 +
<br>*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c
 +
 
 +
 
 +
<h3>Ligation of Test </h3>
 +
 
 +
<b>Procedure</b>
 +
<br>*Mix well and spin down briefly.
 +
<br>*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample  (4ul for sample A, 4 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
 +
<br>*Incubate 1 hour at 22°C.
 +
<br>*inactivation of T4 DNA ligase at 65°C for 10 min
 +
<br>*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
 +
 
 +
<b><br>Notes:</b> T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
 +
 
 +
<br>Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants
 +
 
 +
 
 +
<h3>16 july</h3>
 +
<h4>Transformation of E.coli with </h4>
 +
 
 +
<b>Procedure:</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*3 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>*The tube was placed in ice for 2-5 minutes
 +
<br>*900 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 
 +
<b><br>Notes:</b>
 +
<h3>17 july</h3>
 +
<h4>Last day of summer work! </h4>
 +
<b><br>Notes:</b>Colonies for ligation never appear, all the minipreps were stored at -4°C to continue working on August! 
-
<ul>
 
-
              <li>Ampicilin Stock:</li>
 
-
            </ul>     
 
-
    <p><ol>
 
-
  <li>Weight 100 mg of liophylized antibiotic for each ml of stock solution.</li>
 
-
          <li>Dissolve in distilled water and mix gently (It is possible to use Vortex).</li>
 
-
          <li>For each 2 ml of media use 1 ul of stock antibiotic.</li>
 
-
      </ol></p>
 
-
<p><ol>Therefore the working concentration for each antibiotic (for use in and Agrobacterium tumefaciens and E. coli) will be:
 
-
  <li>Cloramphenicol: 5ul/ml</li>
 
-
          <li>Kanamicin: 25 ul/ml</li>
 
-
          <li>Ampicilin: 50 ul/ml</li>
 
-
      </ol></p>
 
     </div>
     </div>
 +
<div id="prot3" class="protocolactive">
 +
 +
 +
<h3>18 august </h3>
 +
 +
<h4>Media preparation LB broth and LB agar and E.coli k.12, E.coli Top10 and agrobacterium inoculation </h4>
 +
 +
<b>Procedure: </b>
 +
 +
<br>*14g of LB agar were weighted to prepare 400ml of medium
 +
<br>*Weight 42.5 grams of Terrific Broth for each liter of media.
 +
<br>*Autoclave
 +
<br>*E.coli was inoculated in petri dishes and incubated at 37°C
 +
<b><br>Notes:</b>The growth mediums in storage were review; they didn’t show signs of contamination
 +
 +
<h3>19 august </h3>
 +
<h4>Electrophoresis gel of summer minipreps </h4>
 +
 +
<img src="https://static.igem.org/mediawiki/2014/2/2a/19ag.jpg" widht="30" height="400"/>
 +
 +
 +
<b><br>Notes:</b>Most of the DNA from summer minipreps was loses. Probably, due to wrong storage conditions.
 +
<h3>20 august </h3>
 +
<h4>Transformation of E.coli with NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS</h4>
 +
 +
<b>Procedure:</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*3 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>*The tube was placed in ice for 2-5 minutes
 +
<br>*900 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 +
<h3>21 august</h3>
 +
<h4>Media preparation LB broth and  E.coli Top10 inoculation</h4>
 +
 +
<b>Procedure: </b>
 +
<br>*14g of LB agar were weighted to prepare 400ml of medium
 +
<br>*1 g of LB broth were weighted to prepare 500 ml of medium
 +
<br>*Autoclave at 121°C for 15 minutes
 +
<br>*E.coli was inoculated in petri dishes and incubated at 37°C
 +
<b><br>Notes:</b>The growth mediums in storage were review; they didn’t show signs of contamination
 +
 +
<h3>22 august </h3>
 +
<h4>Calcium chloride competent cells</h4>
 +
 +
<b>Procedure:</b>
 +
<br>*Add 1.5 ml of E.coli in LB broth to Ice tubes
 +
<br>*Centrifuged for 3 minutes at 6000 rpm, two times
 +
<br>*The supernatant was discarded
 +
<br>*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
 +
<br>*Incubated in ice for 20 minutes
 +
<br>*The tubes were centrifuged again for 3 minutes at 6000 rpm
 +
<br>*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
 +
<br>*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c
 +
 +
<h4>Transformation of E.coli with NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS</h4>
 +
 +
<b>Procedure</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*3 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>*The tube was placed in ice for 2-5 minutes
 +
<br>*900 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 +
<h3>25 august </h3>
 +
<h4>Restricking plates with summer transformations in LB agar with antibiotics: NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS, MerE and MerB</h4>
 +
 +
<h3>26 august </h3>
 +
<h4>Inoculation of Transformed colonies of summer transformations in terrific broth </h4>
 +
 +
<b>Procedure:</b>
 +
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
 +
 +
<h3>27 august</h3>
 +
<h4>Miniprep: NLS, PhyB, VP16, PiF6, TetR, MerE, Pif6 and MerB</h4>
 +
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7 (room temperature)
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added (65°C preheated)
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
 +
 +
<h4>Electrophoresis gel of minipreps: NLS, PhyB, VP16, PiF6, TetR, MerE, Pif6 and MerB</h4>
 +
 +
<img src="https://static.igem.org/mediawiki/2014/6/65/Fallido.jpg" widht="30" height="400"/>
 +
 +
<h3>28 august </h3>
 +
<h4>Transformation NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS, MerB and MerE</h4>
 +
<b>Procedure</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*3 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>*The tube was placed in ice for 2-5 minutes
 +
<br>*900 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 +
<h3>29 august </h3>
 +
<h4>Inoculation of Transformed colonies of NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS, MerE and MerB in terrific broth</h4> 
 +
 +
<b>Procedure:</b>
 +
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
 +
 +
 +
</div>
 +
 +
<div id="prot4" class="protocolactive">
 +
 +
<h3>1 september </h3>
 +
<h4>Miniprep: MerB and MerE</h4>
 +
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7 (room temperature)
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added (65°C preheated)
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
 +
 +
<h4>Electrophoresis gel  miniprep: MerE y MerB</h4>
 +
 +
<h3>2 september  </h3>
 +
<h4>PCR</h4>
 +
<h4>Electrophoresis Gel: PCR products </h4>
 +
<h3>3 september  </h3>
 +
<h4>PCR</h4>
 +
<h4>Electrophoresis Gel: PCR products</h4>
 +
<h4>Transformation </h4>
 +
<b>Procedure</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*3 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 45 seconds
 +
<br>*The tube was placed in ice for 2-5 minutes
 +
<br>*900 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 +
<h3>4 september  </h3>
 +
<h4>Inoculation of Transformed colonies of  PhyB, Pif6, AGS, NLS, VP16 and PhyB(2) in terrific broth  </h4>
 +
 +
<b>Procedure:</b>
 +
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
 +
 +
<h3>5 september  </h3>
 +
<h4>Miniprep: PhyB, Pif6, AGS, NLS, VP16 and PhyB(2)</h4>
 +
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7 (room temperature)
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added (65°C preheated)
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
 +
 +
<h4>Electrophoresis gel of Miniprep</h4>
 +
 +
<img src="https://static.igem.org/mediawiki/2014/d/df/5de.jpg" widht="30" height="400"/>
 +
 +
<h3>8 september  </h3>
 +
<h4>E.coli top 10 inoculation </h4>
 +
<b>Procedure:</b>
 +
<h4>Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking </h4>
 +
 +
<h4>Plasmid rehydratation of Agrobacterium vectors from paper filter. </h4>
 +
<h3>9 september  </h3>
 +
<h4>E.coli top10 Electrocompent cells </h4>
 +
<b>Procedure:</b>
 +
 +
<br>*Dilute the cells 100X. 100 ml LB Amp.  Grow at OD600 between 0.6 and 0.8.
 +
<br>*Split the culture into 2x 50 ml falcon tubes, on ice.
 +
<br>*Centrifuge at 4 °C for 10 min at 4000 rpm.
 +
<br>*Wash and combine the cells. Remove the supernatant.
 +
<br>*Resuspend the cells in 2x 25 ml of ice cold water. Combine the volumes
 +
<br>*Wash the cells 2 more times and  entrifuge at 4 °C for 10 min at 4000 rpm.
 +
<br>*Resuspend in 50 ml of ice cold water and Repeat.
 +
<br>*Wash and concentrate the cells and Centrifuge at 4 °C for 10 min at 4000 rpm.
 +
<br>*Resuspend in 1-2 ml of ice cold water and storage at -20°C
 +
<h3>10 september  </h3>
 +
<h4>Electro-Transformation of RBS, VP16, Pif6, Pif6 (2) and TetR</h4>
 +
<b>Procedure:</b>
 +
 +
<br>*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
 +
<br>*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
 +
<br>*******Electroporation:
 +
<br>*******Mode Prokaryotes
 +
<br>*******Voltage (V) 1,700 V
 +
<br>*******Time constant (τ) 5 ms
 +
<br>*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
 +
<br>*Plate on LB plates with Ampicillin.
 +
<h3>11 september  </h3>
 +
<h4>Inoculation of E.coli with RBS, VP16, Pif6, Pif6 (2) and TetR in Terrific broth</h4>
 +
<b>Procedure:</b>
 +
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
 +
 +
<h3>12 september  </h3>
 +
<h4>Miniprep: RBS, VP16, Pif6, Pif6 (2) and TetR</h4>
 +
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7 (room temperature)
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added (65°C preheated)
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
 +
 +
<h4>Electrophoresis Gel of miniprep [RBS, VP16, Pif6, Pif6 (2) and TetR]</h4>
 +
 +
<img src="https://static.igem.org/mediawiki/2014/1/1c/12es.jpg" widht="30" height="400"/>
 +
 +
<h3>13 september </h3>
 +
<h4>Restriction digest of MerB, MerE and Psc1c3</h4>
 +
<b>Procedure:</b>
 +
<br>*Add 9 ul of DNA to be digested, and 6 ul of dH20
 +
<br>*Pipet 2.5ul of NEB Buffer 2 to each tube.
 +
<br>*Pipet 0.5ul of BSA to each tube.
 +
<br>*In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of PstI.
 +
<br>*In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI,
 +
<br>*Incubate the restriction digests at 37°C for 60 minutes
 +
 +
<h4>Electrophoresis gel:  Restriction digest of MerB, MerE</h4>
 +
 +
<h3>14 september  </h3>
 +
<h4>Restriction digest of MerB, MerE and Psc1c3 [NEW ENZYMES]</h4>
 +
<b>Procedure:</b>
 +
<br>*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min
 +
 +
<b><br>Notes:</b>
 +
 +
<h4>Electrophoresis gel:  Restriction digest of MerB, MerE</h4>
 +
 +
<img src="https://static.igem.org/mediawiki/2014/f/fb/Es.jpg" widht="30" height="400"/>
 +
 +
<h4>DNA Gel extraction</h4>
 +
<b>Procedure</b>
 +
<br>*Place 400 mg of the excised gel into a 1.5-mL tube.
 +
<br>*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
 +
<br>*Place the tube into a 50°C water bath and at least 10 minutes.
 +
<br>*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
 +
<br>*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
 +
<br>*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
 +
<br>*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
 +
<br>*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
 +
<br>*Store the purified DNA at 4°C
 +
<h3>15 september  </h3>
 +
<h4>Nanodrop</h4>
 +
<h3>Ligation of MerB and MerE in Psb1c3</h3>
 +
<h3>E. coli Electrocompetent cells</h3>
 +
<b>Procedure:</b>
 +
 +
<br>*Dilute the cells 100X. 100 ml LB Amp.  Grow at OD600 between 0.6 and 0.8.
 +
<br>*Split the culture into 2x 50 ml falcon tubes, on ice.
 +
<br>*Centrifuge at 4 °C for 10 min at 4000 rpm.
 +
<br>*Wash and combine the cells. Remove the supernatant.
 +
<br>*Resuspend the cells in 2x 25 ml of ice cold water. Combine the volumes
 +
<br>*Wash the cells 2 more times and  entrifuge at 4 °C for 10 min at 4000 rpm.
 +
<br>*Resuspend in 50 ml of ice cold water and Repeat.
 +
<br>*Wash and concentrate the cells and Centrifuge at 4 °C for 10 min at 4000 rpm.
 +
<br>*Resuspend in 1-2 ml of ice cold water and storage at -20°C
 +
<h3>16 september </h3>
 +
<h4>Electrotransformation of MerB and MerE in Psb1c3</h4>
 +
<b>Procedure:</b>
 +
 +
<br>*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
 +
<br>*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
 +
<br>*******Electroporation:
 +
<br>*******Mode Prokaryotes
 +
<br>*******Voltage (V) 1,700 V
 +
<br>*******Time constant (τ) 5 ms
 +
<br>*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
 +
<br>*Plate on LB plates with Ampicillin.
 +
<h3>17 september </h3>
 +
<h4>Electrotransformation of MerB and MerE in Psb1c3</h4>
 +
<b>Procedure:</b>
 +
 +
<br>*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
 +
<br>*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
 +
<br>*******Electroporation:
 +
<br>*******Mode Prokaryotes
 +
<br>*******Voltage (V) 1,700 V
 +
<br>*******Time constant (τ) 5 ms
 +
<br>*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
 +
<br>*Plate on LB plates with Chloramphenicol.
 +
 +
<h3>18 september  </h3>
 +
<h4>Inoculation in Terrific broth [MerB and MerE in Psb1c3]</h4>
 +
 +
<b>Procedure:</b>
 +
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
 +
 +
 +
<h3>19 september  </h3>
 +
<h4>Miniprep: MerB and MerE in Psb1c3 </h4>
 +
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7 (room temperature)
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added (65°C preheated)
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
 +
 +
<h4>Electrophoresis gel [Miniprep: MerB and MerE in Psb1c3]</h4>
 +
<h3>20 september </h3>
 +
<h4>Creating tools: </h4>
 +
<h4>Media preparation LB broth and LB agar </h4>
 +
 +
<b>Procedure: </b>
 +
<br>*14g of LB agar were weighted to prepare 400ml of medium
 +
<br>*1 g of LB broth were weighted to prepare 500 ml of medium
 +
<br>*Autoclave at 121°C for 15 minutes
 +
<br>*E.coli was inoculated in petri dishes and incubated at 37°C
 +
<h4>Stocks: Ampicillin, Kanamycin and Chloramphenicol</h4>
 +
<b>Procedure:</b>
 +
 +
<br>*******Chloramphenicol Stock:
 +
<br>*Weight 10 mg of lyophilized antibiotic for each ml of stock solution.
 +
<br>*Dissolve in ethanol and mix gently (It is possible to use Vortex)
 +
<br>*******Kanamicin Stock:
 +
<br>*Weight 50 mg of lyophilized antibiotic for each ml of stock solution.
 +
<br>*Dissolve in distilled water and mix gently (It is possible to use Vortex).
 +
<br>*******Ampicilin Stock:
 +
<br>*Weight 100 mg of liophylized antibiotic for each ml of stock solution.
 +
<br>*Dissolve in distilled water and mix gently (It is possible to use Vortex).
 +
<br>*******Working concentration for each antibiotic will be:
 +
<br>*Cloramphenicol: 5l/ml
 +
<br>*Kanamicin: 25 l/ml
 +
<br>*Ampicilin: 50 l/ml
 +
<h4>Inoculation E.coli in Terrific broth</h4>
 +
 +
<b>Procedure:</b>
 +
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
 +
 +
<h3>21 september</h3> 
 +
<h4>E. coli Calcium Chloride competent cell protocol </h4>
 +
<br>*Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning and shake @ 37°C for 3hrs.
 +
<br>*Put the cells on ice for 10 min and collect the cells by centrifugation for 3 mins @6krpm
 +
<br>*Decant supernatant and gently resuspend on 10 mL cold 0.1M CaCl
 +
<br>*Incubate on ice x 20 mins and  centrifuge 3 mins @6krpm
 +
<br>*Discard supernatant and gently resuspend on 5mL cold 0.1MCaCl/15%Glycerol
 +
<br>*Dispense in microtubes (300μL/tube). Freeze in -80°C.
 +
<h4>Transformation: Measure competent cells Transformation efficiency with PGLO</h4>
 +
<h3>22 september  </h3>
 +
<h4>Restriction digest RBS(A), PhyB(B), AGS(A), VP16(B), MerE(C), MerB(C), NLS (A) and PolyA(B)</h4>
 +
<b>Procedure:</b>
 +
 +
<br>*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min
 +
 +
<h4>Electrophoresis gel of restriction digest: RBS, PhyB, AGS, VP16, MerE, MerB, NLS and PolyA</h4>
 +
<h4>DNA Gel extraction </h4>
 +
<b>Procedure</b>
 +
 +
<br>*Place 400 mg of the excised gel into a 1.5-mL tube.
 +
<br>*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
 +
<br>*Place the tube into a 50°C water bath and at least 10 minutes.
 +
<br>*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
 +
<br>*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
 +
<br>*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
 +
<br>*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
 +
<br>*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
 +
<br>*Store the purified DNA at 4°C
 +
<h3>23 september </h3>
 +
<h4>Ligation RBS with PhyB, AGS with VP16, RBS with MerE, RBS with MerB and NLS with PolyA</h4>
 +
<b>Procedure</b>
 +
<br>*Mix well and spin down briefly.
 +
<br>*Prepare a mixture with 12 ul of DNA sample  (6ul for sample A, 6 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of nuclease free water.
 +
<br>*Incubate 1 hour at 22°C. (termocycle)
 +
<br>*inactivation of T4 DNA ligase at 65°C for 10 min
 +
<br>*Use 10 μL of the mixture for transformation of 50 μL of chemically competent cells
 +
 +
<h4>Electrotransformation of E.coli with [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA]</h4>
 +
<b>Procedure:</b>
 +
 +
<br>*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
 +
<br>*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
 +
<br>********Electroporation:
 +
<br>********Mode Prokaryotes
 +
<br>********Voltage (V) 1,700 V
 +
<br>********Time constant (τ) 5 ms
 +
<br>*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
 +
<br>*Plate on LB plates.
 +
<h3>24 september  </h3>
 +
<h4>Inoculation of E.coli with [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA] in Terrific broth </h4>
 +
<b>Procedure:</b>
 +
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
 +
 +
<h3>25 september </h3>
 +
<h4>Miniprep [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA]</h4>
 +
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7 (room temperature)
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added (65°C preheated)
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
 +
 +
<h4>Electrophoresis gel of Miniprep: [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA]</h4>
 +
<h3>26 september  </h3>
 +
<h4>Restriction digest [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and V3]</h4>
 +
<b>Procedure:</b>
 +
<br>*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min
 +
 +
<h4>Electrophoresis Gel of restriction digest: [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and V3]</h4>
 +
<h4>DNA Gel extraction</h4>
 +
<b>Procedure</b>
 +
 +
<br>*Place 400 mg of the excised gel into a 1.5-mL tube.
 +
<br>*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
 +
<br>*Place the tube into a 50°C water bath and at least 10 minutes.
 +
<br>*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
 +
<br>*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
 +
<br>*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
 +
<br>*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
 +
<br>*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
 +
<br>*Store the purified DNA at 4°C
 +
Ligation of RBS-MerE with RBS-MerB,  RBS-PhyB with AGS-VP16 and of RBS-MerE with RBS-MerB in V3.
 +
Procedure
 +
<br>*Mix well and spin down briefly.
 +
<br>*Prepare a mixture with 12 ul of DNA sample  (6ul for sample A, 6 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of nuclease free water.
 +
<br>*Incubate 1 hour at 22°C. (termocycle)
 +
<br>*inactivation of T4 DNA ligase at 65°C for 10 min
 +
<br>*Use 10 μL of the mixture for transformation of 50 μL of chemically competent cells
 +
 +
<h4>Transformation of E.coli and Agrobacterium with [RBS-MerE-RBS-MerB and RBS-PhyB-AGS-VP16]</h4>
 +
<b>Procedure:</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*10 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 60 seconds
 +
<br>*The tube was placed in ice for 5 minutes
 +
<br>*200 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 2 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 +
<h3>27 september  </h3>
 +
<h4>Inoculation of E.coli and Agrobacterium with [RBS-MerE-RBS-MerB] in Terrific broth  </h4>
 +
<b>Procedure:</b>
 +
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
 +
 +
<h3>28 september</h3> 
 +
<h4>Miniprep [RBS-MerE-RBS-MerB]</h4>
 +
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7 (room temperature)
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added (65°C preheated)
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
 +
 +
<h4>Electrophoresis gel of Miniprep: [RBS-MerE-RBS-MerB]</h4>
 +
<h3>29 september  </h3>
 +
<h4>Restriction digest of RBS-PhyB and AGS-VP16 </h4>
 +
<b>Procedure:</b>
 +
 +
<br>*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min
 +
 +
<h4>Electrophoresis Gel of Restriction digest [RBS-PhyB and AGS-VP16]</h4>
 +
<h3>30 september  </h3>
 +
<h4>Restriction digest of RBS-PhyB and AGS-VP16 </h4>
 +
<b>Procedure:</b>
 +
<br>*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min</br>
 +
 +
<h4>Electrophoresis Gel of Restriction digest [RBS-PhyB and AGS-VP16]</h4>
 +
<h4>DNA gel extraction</h4>
 +
<b>Procedure</b>
 +
 +
<br>*Place 400 mg of the excised gel into a 1.5-mL tube.
 +
<br>*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
 +
<br>*Place the tube into a 50°C water bath and at least 10 minutes.
 +
<br>*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
 +
<br>*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
 +
<br>*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
 +
<br>*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
 +
<br>*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
 +
<br>*Store the purified DNA at 4°C
-
    <div id="prot4" class="protocol">
 
-
      <h2> CaCl2 Competent Cells </h2>
 
-
      <p>
 
-
<ol>
 
-
  <li> Streak out frozen glycerol stock of bacterial cells (Top10, DH5α, etc.) onto an LB plate (no antibiotics since these cells do not have a plasmid in them). Work sterile. Grow plate overnight at 37°C.
 
-
  </li>
 
-
  <li>Make sure to autoclave 1 L LB (or your preferred media), 1 L of 100 mM CaCl2, 1 L of 100 mM MgCl2, 100 mL of 85 mM CaCl2, 15% glycerol v/v, 4 centrifuge bottles and caps, lots of microfuge tubes
 
-
  </li>
 
-
  <li>Chill overnight at 4°C 100 mM CaCl2, 100 mM MgCl2, 85 mM CaCl2, 15% glycerol v/v  </li>
 
-
  <li>Prepare starter culture of cells  </li>
 
-
  <li>Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter culture of LB (or your preferred media – no antibiotics). Grow culture at 37°C in shaker overnight.
 
-
  </li>
 
-
  <li>For the next day, inoculate 1 L of LB media with 10 mL starter culture and grow in 37°C shaker.
 
-
  </li>
 
-
<li>Measure the OD600 every hour, then every 15-20 minutes when the OD getsabove 0.2.
 
-
  </li>
 
-
<li>When the OD600 reaches 0.35-0.4, immediately put the cells on ice. Chill the culture for 20-30 minutes, swirling occasionally to ensure even cooling. Place centrifuge bottles on ice at this time.
 
-
  </li>
 
-
<li>(Spin #1) Split the 1 L culture into four parts by pouring about 250 mL into ice cold centrifuge bottles. Harvest the cells by centrifugation at 3000g for 15 minutes at 4°C.
 
-
  </li>
 
-
<li>Decant the supernatant and gently resuspend each pellet in about 100 mL of ice cold MgCl2. Combine all suspensions into one centrifuge bottle. Make sure to prepare a blank bottle as a balance.  </li>
 
-
<li>(Spin #2) Harvest the cells by centrifugation at 2000g in the refrigerated centrifuge (~3000 rpm) for 15 minutes at 4°C.  </li>
 
-
<li>Decant the supernatant and resuspend the pellet in about 200 mL of ice cold CaCl2. Keep this suspension on ice for at least 20 minutes. Start putting 1.5 mL microfuge tubes on ice if not already chilled.  </li>
 
-
<li>(Spin #3) Harvest the cells by centrifugation at 2000g (~3000 rpm) for 15 minutes at 4°C. At this step, rinse a 50 mL conical tube with ddH2O and chill on ice.  </li>
 
-
<li>Decant the supernatant and resuspend the pellet in ~50 mL of ice cold 85 mM CaCl2, 15% glycerol. Transfer the suspension to the 50 mL conical tube.  </li>
 
-
<li>(Spin #4) Harvest the cells by centrifugation at 1000g (~2100) for 15 minutes at 4°C.  </li>
 
-
<li>Decant the supernatant and resuspend the pellet in 2 mL of ice cold 85 mM CaCl2, 15% glycerol. The final OD600 of the suspended cells should be ~200-250.  </li>
 
-
<li>Aliquot 50 μL into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer. </li>
 
-
</ol>
 
       </div>
       </div>
-
    <div id="prot5" class="protocol">
+
<div id="prot5" class="protocolactive">
-
      <h2> Heat Shock Transformation of E. coli </h2>
+
<h3>1 october  </h3>
-
      <p><b>Note:</b> Never vortex competent cells. Mix cells by gentle shaking.
+
<h4>Ligation of RBS-PhyB with AGS-VP16</h4>
-
<ol>
+
<b>Procedure</b>
-
  <li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.
+
<br>*Mix well and spin down briefly.
-
  </li>
+
<br>*Prepare a mixture with 12 ul of DNA sample  (6ul for sample A, 6 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of nuclease free water.  
-
  <li>Place 20 ul of cells in a pre-chilled Eppendorf tube.
+
<br>*Incubate 1 hour at 22°C. (termocycle)
-
<ul>
+
<br>*inactivation of T4 DNA ligase at 65°C for 10 min
-
              <li><u>For an Intact Vector:</u> Add 0.5 ul or less to the chilled cells</li>
+
<br>*Use 10 μL of the mixture for transformation of 50 μL of chemically competent cells  
-
              <li><u>For a Ligation Product:</u> Add 2-3 ul to the chilled cells.</li>
+
-
    </ul>
+
-
  </li>
+
-
  <li>Mix gently by flicking the tube.
+
-
  </li>
+
-
  <li>Chill on ice for 10 minutes. (Optional)
+
-
  </li>
+
-
  <li>Heat shock at 42 °C for 50 seconds.
+
-
  </li>
+
-
  <li>Incubate on ice for 2 minutes.
+
-
  </li>
+
-
  <li>Add 200 ul LB, Terrific or SOC medium and recover the cells by shaking at 37 °C.  
+
-
  </li>
+
-
  <li>The recovery time varies with the antibiotic selection.  
+
-
<ul>
+
-
              <li>Ampicillin: 15-30 minutes</li>
+
-
              <li>Kanamycin or Spectinomycin: 30-60 minutes</li>
+
-
              <li>Chloramphenicol: 60-120 minutes</li>
+
-
              </ul>
+
-
  </li>
+
-
  <li>Plate out the cells on selective LB. Use glass beads to spread the cells. The volume of cells plated depends on what is being transformed.
+
-
<ul>
+
-
              <li><u>For an Intact Vector:</u> High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.</li>
+
-
              <li><u>For a Ligation Product:</u> Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.</li>
+
-
    </ul>
+
-
  </li>
+
-
  <li>Incubate at 37 °C. Transformants should appear within 8 – 16 hrs.
+
-
  </li>
+
-
 
+
-
</ol>
+
-
      </p>
+
-
    </div>
+
-
    <div id="prot6" class="protocol">
+
<h4>Transformation of E.coli with [RBS-PhyB-AGS-VP16]</h4>  
-
      <h2>Transformation by Electroporation </h2>
+
<b>Procedure:</b>
-
      <h3>Preparation of Electrocompetent Cells</h3>
+
<br>*50 µl of competent cells were placed in a cold eppendorf tube
-
      <p>Competent cells should never be vortexted, as this will cause them to lyse and release salts into the media. Resuspend cells by pipeting up and down with a large pasteur pipet. Once they are chilled, cells should be continuously cold.
+
<br>*10 µl of DNA sample were added
-
<ol>
+
<br>*It was mixed gently and left incubating in ice for 30 minutes
-
  <li>The night before the transformation, start an overnight culture of cells. 5 ml LB Amp.</li>
+
<br>*The tube was put through heat shock in water at 42⁰c for 60 seconds
-
  <li>The day of the transformation, dilute the cells 100X. 100 ml LB Amp. </li>
+
<br>*The tube was placed in ice for 5 minutes
-
  <li>Grow at 30°C for about 90 minutes.</li>
+
<br>*200 µl of S.O.C. were added
-
  <li>Harvest the cells. When the cells reach an OD600 of between 0.6 and 0.8. </li>
+
<br>*The tube was left incubating for 2 hour at 37⁰c and 120 rpm
-
  <li>Split the culture into 2x 50 ml falcon tubes, on ice. </li>
+
<br>*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
-
  <li>Centrifuge at 4 °C for 10 min at 4000 rpm.</li>
+
<br>*The dish was sealed and left incubating at 37⁰c
-
  <li>Wash and combine the cells.</li>
+
-
  <li>Remove the supernatant.</li>
+
-
  <li>Resuspend the cells in 2x 25 ml of ice cold water. </li>
+
-
  <li>Combine the volumes in a single 50 ml falcon tube. </li>
+
-
  <li>Wash the cells 2 more times. </li>
+
-
  <li>Centrifuge at 4 °C for 10 min at 4000 rpm.</li>
+
-
  <li>Resuspend in 50 ml of ice cold water.</li>
+
-
  <li>Repeat.</li>
+
-
  <li>Wash and concentrate the cells for electroporation.</li>
+
-
  <li>Centrifuge at 4 °C for 10 min at 4000 rpm.</li>
+
-
  <li>Resuspend in 1-2 ml of ice cold water.</li>
+
-
          <li>We will use 200 ul of washed cells per transformation.</li>
+
-
</ol>
+
-
      <h3>Dialysis of PCR or Digestion Products</h3>
+
-
        <ol>
+
-
  <li>DNA for electroporation must be free of salts to avoid arcing.
+
-
  </li>
+
-
  <li>Float a filter in a Petri dish filled with water.
+
-
  </li>
+
-
  <li>Millipore membrane filter 0.025 uM.
+
-
  </li>
+
-
  <li>Pipet one drop of PCR product onto the filter.
+
-
  </li>
+
-
  <li>200 ng is needed per transformation.
+
-
  </li>
+
-
  <li>20 - 100 ul fits well on one filter.
+
-
  </li>
+
-
  <li>Collect the drop after 30 - 45 minutes.
+
-
          </li>
+
-
  <li>The volume will change, but the DNA is not lost.
+
-
          </li>
+
-
</ol>
+
-
      </p>
+
-
    </div>
+
-
   
+
-
    <div id="prot7" class="protocol">
+
-
      <h2>Plasmid purification by Miniprep </h2>
+
-
      <p>
+
-
      <ol>
+
-
  <li>Centrifuge an eppendorf tube of 1.5ml with L. Plantarum culture at 12000 rpm in the mini-spin for 1 minute, two times.
+
-
  </li>
+
-
  <li>Discard the supernatant and resuspend the tube’s content with more L. Plantarum culture in MRS broth.
+
-
  </li>
+
-
  <li>Centrifuge the eppendorf tube for 15min at 6000 rpm.
+
-
  </li>
+
-
  <li>Discard the supernatant.
+
-
  </li>
+
-
  <li>Resuspend the pellet in 250ml Resuspension buffer.
+
-
  </li>
+
-
  <li>Add 250ml of Lysis Buffer.
+
-
  </li>
+
-
  <li>Gently mix the tube carefully inverting it 5 times carefully.
+
-
  </li>
+
-
  <li>Add and mix softly 350ml of Precipitation Buffer, inverting the tube.
+
-
  </li>
+
-
  <li>Centrifuge it at 12000 rpm for 10 minutes.
+
-
  </li>
+
-
  <li>Transfer the supernatant into a spin column inside a washtube.
+
-
  </li>
+
-
  <li>Centrifuge it at 12000 rpm for a minute.
+
-
  </li>
+
-
  <li>Discard the supernatant and add 500 ml of Wash Buffer with ethanol (w10) to the column.
+
-
  </li>
+
-
  <li>Incubate it for one minute at room temperature.
+
-
  </li>
+
-
  <li>Centrifuge the column at 12000 rpm for 1 minute.
+
-
  </li>
+
-
  <li>Discard the liquid from the washtube and place the column inside the tube.
+
-
  </li>
+
-
  <li>Add 700ml of Wash Buffer W9 with ethanol to the column.
+
-
  </li>
+
-
  <li>Centrifuge the column with the washtube at 12000 rpm for 1 minute.
+
-
  </li>
+
-
  <li>Discard the liquid from the washtube.
+
-
  </li>
+
-
  <li>Centrifuge the column with the washtube at 12000 rpm for 1 minute.
+
-
  </li>
+
-
  <li>Discard the liquid from the washtube.
+
-
  </li>
+
-
  <li>Place the column inside an eppendorf tube of 1.5ml.
+
-
  </li>
+
-
  <li>Add 75ml of preheated TE Buffer at the center of the column.
+
-
  </li>
+
-
  <li>(Warm the TE Buffer previously in water bath at 65⁰c-70⁰c for 3 minutes).
+
-
  </li>
+
-
  <li>Incubate the column for 1 minute at room temperature.
+
-
  </li>
+
-
  <li>The column was centrifuged at 12000 rpm for 2 minutes.
+
-
  </li>
+
-
  <li>(The eppendorf tube contains the purified plasmid).
+
-
  </li>
+
-
</ol>
+
-
      </p>
+
-
    </div>
+
-
    <div id="prot8" class="protocol">
+
<h3>2 october </h3>  
-
      <h2>Plasmid purification by Midiprep</h2>
+
<h4>Inoculation of E.coli with [RBS-PhyB-AGS-VP16] in Terrific broth </h4>
-
<p>
+
<b>Procedure:</b>
-
      <ol>
+
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
-
  <li>Harvest the cells by centrifugation for 10 min at 12000 rpm in the mini-spin. Discard the supernatant.</li>
+
-
  <li>Resuspend the pelleted cells in 2 mL of Resuspension Solution auditioned with RNase solution. The bacterial pellet should be resuspended by vortexing or pipetting up and down until no cell clumps remain.
+
-
  </li>
+
-
  <li>Add 2 mL of Lysis Solution and mix gently by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Incubate for 3 min at room temperature. </li>
+
-
  <li>Add 0.5 mL of the Endotoxin Binding Reagent. Mix immediately by inverting the tube 5-8 times.
+
-
  </li>
+
-
  <li>Incubate for 5 min at room temperature.  <b>Note.</b> After the addition of the Neutralization Solution and Endotoxin Binding Reagent it is important to mix gently, but thoroughly, to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should appear cloudy and contain white precipitate. </li>
+
-
  <li>Add 3 mL of 96% ethanol. Mix immediately by inverting the tube 5-6 times.
+
-
  </li>
+
-
  <li>Centrifuge for 10 min at 4,000-5,000 rpm to pellet cell debris and chromosomal DNA.</li>
+
-
  <li>Transfer the supernatant to a 15 mL tube (not provided) by decanting or pipetting. Avoid disturbing or transferring the white precipitate. </li>
+
-
  <li>Add 3 mL of 96% ethanol. Mix immediately by inverting the tube 5-6 times. </li>
+
-
  <li>Transfer part of the sample (~ 5.5 mL) to the supplied column pre-assembled with a collection tube (15 mL). Be careful not to overfill the column. Centrifuge for 3 min 10000 rpm in the mini-spin. Discard the flow-through and place the column back into the same collection tube. </li>
+
-
  <li>Repeat the last step to process any remaining lysate through the purification column.</li>
+
-
  <li>Add 4 mL of Wash Solution I (diluted with isopropanol) to the purification column. Centrifuge for 2 min. at 4000 rpm in a swinging bucket rotor. Discard the flow-through and place the column back into the same collection tube. </li>
+
-
  <li>Add 4 mL of Wash Solution II (diluted with ethanol) to the purification 6 column. Centrifuge for 2 min. at 5000 rpm. Discard the flow-through and place the column back into the same collection tube. </li>
+
-
  <li>Repeat the column wash with Wash Solution II</li>
+
-
  <li>Centrifuge for 5 min at 3,000 × g in a swinging bucket rotor to remove residual wash solution. Discard the collection tube containing the flow-through.</li>
+
-
  <li>Transfer the column into a fresh 15 mL collection tube (provided). Add 0.35 mL of the Elution Buffer to the centre of the purification column membrane. Incubate for 2 min at room temperature and centrifuge for 5 min at 5000 rpm to elute plasmid DNA. </li>
+
-
  <li>Discard the purification column. Use the purified plasmid DNA in downstream applications or store DNA at -20°C. </li>
+
-
</ol>
+
<h3>3 october  </h3>
-
      </p>
+
<h4>Miniprep [RBS-PhyB-AGS-VP16]</h4>
-
    </div>
+
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7 (room temperature)
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added (65°C preheated)
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
-
    <div id="prot9" class="protocol">
+
<h4>Electrophoresis gel of Miniprep: [RBS-PhyB-AGS-VP16]</h4>
-
      <h2>Electrophoresis Agarose Gel</h2>
+
-
      <p>
+
-
<ol>
+
-
  <li>For each 100 ml of electrophoresis gel, weight 1 g of agarose.</li>
+
-
  <li>Dissolve in the adequate amount of 1x TAE Buffer.</li>
+
-
  <li>Heat in constant agitation. It could be done in microwave by heating in short intervals and agitating manually. </li>
+
-
  <li>When completely dissolved let the flask cool a little then and 1.2 l of ethidium bromide 5 % p/p for each 50 ml of gel.</li>
+
-
  <li>Agitate until it’s completely dissolved. If not used immediately store at 4 °C.</li>
+
-
  <li>Pour the agarose into a gel tray with the suitable well comb in place (pour slowly to avoid bubbles which will disrupt the gel).</li>
+
-
  <li>Place newly poured gel at room temperature for 20-30 minutes, until the gel has completely solidified.</li>
+
-
  <li>Once solidified, remove the comb and place the gel into the electrophoresis unit (gel box).</li>
+
-
  <li>Fill the gel box with 1x TAE buffer.</li>
+
-
  <li>Load GeneRuler 1Kb Plus DNA Ladder 0.1 ug/ml weight one lane of the gel.</li>
+
-
  <li>Carefully load your samples into the additional wells of the gel, carefully mixed with 6X DNA Loading Dye.</li>
+
-
  <li>Run the gel at 50-150V until the dye line is approximately 75-80% of the way down the gel (20-45 min).</li>
+
-
  <li>Use an UV light transilluminator to observe the DNA fragments on the gel. </li>
+
-
</ol>
+
-
      </p>
+
-
    </div>
+
-
    <div id="prot10" class="protocol">   
 
-
      <h2>Restriction Digest</h2>
 
-
      <ol>
 
-
  <li>Keep all enzymes and buffers used on ice.</li>
 
-
  <li>Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes, and flick/spin them to collect the liquid at the bottom of the tube.</li>
 
-
  <li>Add 250ng of DNA to the appropriately labelled tube. Add distilled water to the tubes for a total volume of 16ul in each tube.</li>
 
-
  <li>Pipet 2.5ul of NEB Buffer 2 to each tube.</li>
 
-
  <li>Pipet 0.5ul of BSA to each tube.</li>
 
-
  <li>In the <b>Part A</b> tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.</li>
 
-
  <li>In the <b>Part B</b> tube: Add 0.5ul of XbaI, and 0.5ul of PstI.</li>
 
-
  <li>In the <b>pSB1C3</b> tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.</li>
 
-
  <li>The total volume in each tube should be approximately 20ul. Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture to the bottom of the tube.</li>
 
-
  <li>Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes. We use a thermal cycler with a heated lid.</li>
 
-
  <li>Use ~2ul of the digest (25ng of DNA) for ligations.</li>
 
-
</ol>
 
-
<style type="text/css">
 
-
.tftable {font-size:12px;color:#333333;width:100%;border-width: 1px;border-color: #ebab3a;border-collapse: collapse;}
 
-
.tftable th {font-size:12px;background-color:#e6983b;border-width: 1px;padding: 8px;border-style: solid;border-color: #ebab3a;text-align:left;}
 
-
.tftable tr {background-color:#ffffff;}
 
-
.tftable td {font-size:12px;border-width: 1px;padding: 8px;border-style: solid;border-color: #ebab3a;}
 
-
.tftable tr:hover {background-color:#ffff99;}
 
-
</style>
 
-
<table class="tftable" border="1">
+
<h3>4 october</h3>
-
<tr><th></th><th>Part A</th><th>Part B</th><th>linearized plasmid backbone</th></tr>
+
<h4>Restriction digest of RBS-PhyB-AGS-VP16 and NLS-PolyA</h4>
-
<tr><td>DNA</td><td>250ng</td><td>250ng</td><td>250ng (10ul @ 25ng/ul)</td></tr>
+
<b>Procedure:</b>
-
<tr><td>dH2O</td><td>adjust to 16ul</td><td>adjust to 16ul</td><td>6ul</td></tr>
+
-
<tr><td>NEB Buffer 2</td><td>2.5ul</td><td>2.5ul</td><td>2.5ul</td></tr>
+
-
<tr><td>BSA</td><td>0.5ul</td><td>0.5ul</td><td>0.5ul</td></tr>
+
-
<tr><td>Enzyme 1</td><td>0.5ul EcoRI</td><td>0.5ul Xbal</td><td>0.5ul EcoRI</td></tr>
+
-
<tr><td>Enzyme 2 Cell:1</td><td>0.5ul SpeI</td><td>0.5ul PstI</td><td>0.5ul Pst1</td></tr>
+
-
<tr><td>Enzyme 3</td><td></td><td></td><td>0.5ul Dpnl</td></tr>
+
-
</table>
+
-
      </p>
+
-
    </div>
+
-
   
+
-
 
+
-
    <div id="prot11" class="protocol">   
+
-
      <h2>Parts Ligation</h2>
+
-
      <ol>
+
-
  <li>Sticky-end ligation.</li>
+
-
  <li>Prepare the following reaction mixture: </li>
+
-
<style type="text/css">
+
<br>*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min
-
.tftable {font-size:12px;color:#333333;width:100%;border-width: 1px;border-color: #ebab3a;border-collapse: collapse;}
+
-
.tftable th {font-size:12px;background-color:#e6983b;border-width: 1px;padding: 8px;border-style: solid;border-color: #ebab3a;text-align:left;}
+
-
.tftable tr {background-color:#ffffff;}
+
-
.tftable td {font-size:12px;border-width: 1px;padding: 8px;border-style: solid;border-color: #ebab3a;}
+
-
.tftable tr:hover {background-color:#ffff99;}
+
-
</style>
+
-
<table class="tftable" border="1">
+
<b><br>Notes:</b>
-
<tr><th>Linear Vector DNA</th><th>20 - 100 ng</th></tr>
+
-
<tr><td>Insert DNA</td><td>1:1 to 5:1 molar ratio over vector</td></tr>
+
-
<tr><td>10X T4 DNA Ligase Buffer</td><td>2 µl</td></tr>
+
-
<tr><td>T4 DNA Ligase</td><td>1 u</td></tr>
+
-
<tr><td>Nuclease-free water</td><td>To 20 µl</td></tr>
+
-
<tr><td>Total volume</td><td>20 µl</td></tr>
+
-
</table>
+
-
          <li>Incubate 10 min at 22°C.</li>
+
<h4>Electrophoresis gel of Restriction digest [RBS-PhyB-AGS-VP16 and NLS-PolyA]</h4>
-
          <li>Use up to 5 µl of the mixture for transformation of 50 µl of chemically competent cells or 1-2 µl per 50 µl </li>
+
<h4>DNA Gel purification </h4>
-
          <li>The electrotransformation efficiency may be improved by heat inactivation of T4 DNA ligase at 65°C for 10 min or at 70°C for 5 min</li>
+
<b>Procedure</b>
-
          <li>The overall number of transformants may be increased by extending the reaction time to 1 hour. </li>
+
-
          <li>If more than 2 u of T4 DNA ligase is used in 20 μl reaction mixture, it is necessary to purify DNA (by spin column or chloroform extraction) before electrotransformation.</li>
+
-
</ol>
+
<br>*Place 400 mg of the excised gel into a 1.5-mL tube.
 +
<br>*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
 +
<br>*Place the tube into a 50°C water bath and at least 10 minutes.
 +
<br>*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
 +
<br>*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
 +
<br>*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
 +
<br>*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
 +
<br>*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
 +
<br>*Store the purified DNA at 4°C
-
      </p>
 
-
    </div>
 
-
   
 
-
    <div id="prot12" class="protocol">   
 
-
      <h2>Gel Purification</h2>
 
-
      <p>
 
-
<ol>
 
-
  <li>Excise the area of the gel containing your desired DNA fragment using a clean, sharp razor blade. Minimize the amount of agarose surrounding the DNA fragment. </li>
 
-
  <li>Weigh the gel slice containing the DNA fragmen, then place the gel into a 1.5- or 5.0-mL microcentrifuge tube.</li>
 
-
Note: The maximum amount of starting material (gel) is ≤400 mg per tube. If your gel slice exceeds 400 mg, cut the gel into smaller slices so that no one piece exceeds 400 mg. Place each additional gel slice created into separate microcentrifuge tubes.</br>
 
-
  For ≤2% agarose gels:
 
-
</ol>
 
-
        <ol>
 
-
        <li>Place up to 400 mg of the excised gel containing the DNA fragment (previous page) into a 1.5-mL polypropylene microcentrifuge tube. </li>
 
-
        <li>Add 3 volumes Gel Solubilization Buffer for every 1 volume of gel (e.g., add 1.2 mL Gel Solubilization Buffer for a 400 mg gel piece).</li>
 
-
        <li>Place the tube(s) containing your gel slice and Gel Solubilization Buffer into a 50°C water bath or heat block. </li>
 
-
        <li>Incubate the gel slice and Gel Solubilization Buffer at 50°C for at least 10 minutes. Invert the tube by hand every 3 minutes to mix and ensure gel dissolution.</li>
 
-
Note: High concentration gels (>2% agarose) or large gel slices may take longer than 10 minutes to dissolve.
 
-
        <li>After the gel slice appears dissolved, incubate the tube for an additional 5 minutes. </li>
 
-
Optional: For optimal DNA yields, add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
 
-
        <li>Pipet the dissolved gel piece containing the DNA fragment of interest (steps 4–5, page 6) onto the center of a Quick Gel Extraction Column inside a Wash Tube.</li>
 
-
Note: Do not load >400 mg dissolved agarose per Quick Gel Extraction Column.
 
-
        <li>Centrifuge at >12,000 × g for 1 minute. </li>
 
-
        <li>Discard the flow-through and replace the Quick Gel Extraction Column into the Wash Tube. </li>
 
-
        <li>Add 500 μL Wash Buffer (W1), containing ethanol to the Quick Gel Extraction Column. </li>
 
-
        <li>Centrifuge at >12,000 × g for 1 minute. </li>
 
-
        <li>Discard the flow-through and replace the column into the Wash Tube. </li>
 
-
        <li>Centrifuge the column again at maximum speed for 1–2 minutes to remove any residual Wash Buffer and ethanol. Discard the Wash Tube and place the Quick Gel Extraction Column into a Recovery Tube. </li>
 
-
        <li>Add 50 μL Elution Buffer (E5) to the center of the Quick Gel Extraction Column. </li>
 
-
        <li>Incubate the column for 1 minute at room temperature. </li>
 
-
        <li>Centrifuge the Column at >12,000 × g for 1 minute. The Recovery Tube contains the purified DNA. Discard the Quick Gel Extraction Column. </li>
 
-
        <li>Store the purified DNA, or proceed to your downstream application of choice.</li>
 
-
        </ol>
 
-
      </p>
 
-
    </div>
 
-
    <div id="prot13" class="protocol">  
+
<h4>Ligation of RBS-PhyB-AGS-VP16 with NLS-PolyA</h4>
-
      <h2>Gel Electrophoresis</h2>
+
<b>Procedure</b>
-
      <h3>Standard 1% agarose gel</h3>
+
<br>*Mix well and spin down briefly.
-
      <p>Agarose gels are commonly used in concentrations between 0.7 to 2% depending on the size of bands needed to be separated. Simply adjust the amount of
+
<br>*Prepare a mixture with 12 ul of DNA sample  (6ul for sample A, 6 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of nuclease free water.  
-
        starting agarose to %g/100mL TAE e.g. 2g/100mL will give 2%.
+
<br>*Incubate 1 hour at 22°C. (termocycle)
-
<ol>
+
<br>*inactivation of T4 DNA ligase at 65°C for 10 min
-
  <li>Measure out 1g of agarose</li>
+
<br>*Use 10 μL of the mixture for transformation of 50 μL of chemically competent cells
-
  <li>Pour agarose powder into a microwavable flask along with 100mL of 1xTAE</li>
+
-
  <li>Microwave for 1-3mins (until the agarose has dissolved completely and there is a nice rolling boil). Caution HOT! Be careful stirring, eruptive
+
-
        boiling can occur.</li>
+
-
  <li>Let agarose solution cool down for 5min.</li>
+
-
  <li>Pour the agarose into a gel tray with the suitable well comb in place (pour slowly to avoid bubbles which will disrupt the gel).</li>
+
-
  <li>Place newly poured gel at 4°C for 10-15 minutes or let sit at room temperature for 20-30 minutes, until the gel has completely solidified.</li>
+
-
  <li>Once solidified, remove the comb and place the gel into the electrophoresis unit (gel box).</li>
+
-
  <li>Fill the gel box with 1xTAE buffer.</li>
+
-
  <li>Carefully load GeneRuler 100bp Plus DNA weight ladder into the first lane of the gel.</li>
+
-
  <li>Carefully load your samples into the additional wells of the gel.</li>
+
-
          <li>Run the gel at 50-150V until the dye line is approximately 75-80% of the way down the gel.</li>
+
-
  <li>Turn off the power, disconnect the electrodes from the power source and then carefully remove the gel from the gel box.</li>
+
-
  <li>Place the gel into a container filled with 100ml of 1xTAE running buffer and 5μL of EtBr for 5 minutes.</li>
+
-
  <li>Use any device that has UV light to visualize the DNA fragments.</li>
+
-
</ol>
+
-
      </p>
+
-
    </div>
+
-
   
+
 +
<h3>5 october  </h3>
 +
<h4>Transformation of E.coli with [RBS-PhyB-AGS-VP16-NLS-PolyA] </h4>
 +
<b>Procedure:</b>
 +
<br>*50 µl of competent cells were placed in a cold eppendorf tube
 +
<br>*10 µl of DNA sample were added
 +
<br>*It was mixed gently and left incubating in ice for 30 minutes
 +
<br>*The tube was put through heat shock in water at 42⁰c for 60 seconds
 +
<br>*The tube was placed in ice for 5 minutes
 +
<br>*200 µl of S.O.C. were added
 +
<br>*The tube was left incubating for 2 hour at 37⁰c and 120 rpm
 +
<br>*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
 +
<br>*The dish was sealed and left incubating at 37⁰c
 +
 +
<h3>6 october </h3>
 +
<h4>Inoculation of E.coli with [RBS-PhyB-AGS-VP16-NLS-PolyA] in Terrific broth</h4>
 +
<b>Procedure:</b>
 +
<br>*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking
 +
 +
<h3>7 october  </h3>
 +
<h4>Miniprep [RBS-PhyB-AGS-VP16-NLS-PolyA]</h4>
 +
 +
<b>Procedure:</b>
 +
<br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
 +
<br>*Resuspend in 250 µl resuspension buffer
 +
<br>*Add 250 µl of Lysis Buffer L7 (room temperature)
 +
<br>*Add 350 µl of precipitation Buffer N4
 +
<br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
 +
<br>*Centrifuge at 12000 rpm for a minute
 +
<br>*The supernatant was discarded and 500 µl of Wash Buffer were added
 +
<br>*The column was centrifuged at 12000 rpm for 1 minute
 +
<br>*700 µl of Wash Buffer W9 with ethanol were added to the column
 +
<br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute
 +
<br>*75 µl of preheated TE Buffer were added (65°C preheated)
 +
<br>*The column was centrifuged at 12000 rpm for 2 minutes
 +
 +
<h4>Electrophoresis gel of Miniprep: [RBS-PhyB-AGS-VP16-NLS-PolyA]</h4>
 +
<img src="https://static.igem.org/mediawiki/2014/c/c9/Ultima_.jpg" widht="30" height="400"/>
 +
<h3>8 october  </h3>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
   </div>
   </div>
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   <div style="clear: both;"></div>
+
 
 +
   </div>
 +
 
 +
</body>
 +
 
</div>
</div>
</html>
</html>

Latest revision as of 03:57, 18 October 2014

Lab Records

3 June

Team meeting


Notes: Summer lab Schedule!

4 June

Media preparation LB broth and LB agar and E.coli inoculation

Procedure:
* 14g of LB agar were weighted to prepare 400ml of medium
* 1 g of LB broth were weighted to prepare 500 ml of medium
* Autoclave at 121°C for 15 minutes
* E.coli was inoculated in petri dishes and incubated at 37°C
Notes: The growth mediums in storage were revised; they didn’t show signs of contamination

Calcium chloride for competent cells

Procedure:
* Sterilize distilled water in the autoclave for 15 minutes at 121⁰c
* Dilute 1.1159 g of calcium chloride in 50 ml of sterile distilled water
* Fill a syringe to its maximum capacity and connect a ministart 0.2µm filter
* Pour the content of the syringe into a sterile falcon tube through the filter

5 June

E.coli Competent cells

Procedure:
* Add 1.5 ml of E.coli in LB broth to Ice tubes
* Centrifuged for 3 minutes at 6000 rpm, two times
* The supernatant was discarded
* The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
* Incubated in ice for 20 minutes
* The tubes were centrifuged again for 3 minutes at 6000 rpm
* The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
* They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c

MS media preparation for Arabidopsis

Procedure:
* For 500 ml of MS media measure:5 g of sucrose, 2.2 g of basal MS salt, 4 g of agar, 5 ml of Vitamin Gamblor and 500 ml of distilled water
* Mix all the components, except the agar, in constant agitation.
* Adjust the pH to 5.7 ± 0.1.
* Add the agar and mix gently and heat until it reaches 65 – 70 °C. (Do not boil or autoclave)
* Pour the agar in plates, let them cool and store at 4 °C.

6 June

Transformation of E.coli with MerB and MerE (from DNA synthesis)

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*4 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and ampicillin(100mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes:The concentration of lyophilized DNA from synthesis was 200ng, rehydrated with 1 ml of elution buffer, for a final concentration of 2ng/10 ul. E.coli inoculation from SOC media in LB dishes with ampicillin, will be done tomorrow The first colonies appear three days later. The transformation efficiency of competent cells should be review.

9 June

Growing arabidopsis seeds in media

Procedure:
Notes:

Biobricks rehydration and transformation of E.coli: PhyB, Pif6, NLS, VP16 and RFP 10 pg( transformation efficiency)

Procedure:
* Kit plates were rehydrated with 10 ul of distillated water
* 50 µl of competent cells were placed in a cold eppendorf tube
* 2 µl of DNA sample were added
* It was mixed gently and left incubating in ice for 30 minutes
* The tube was put through heat shock in water at 42⁰c for 45 seconds
* The tube was placed in ice for 2-5 minutes
* 900 µl of S.O.C. were added
* The tube was left incubating for 1 hour at 37⁰c and 120 rpm
* A dish with LB agar and Chloramphenicol (35mg/ml) was striated with the transformed cells
* The dish was sealed and left incubating at 37⁰c

Notes:The first colonies appear two days later and transformation efficiency kit is not working as expected. The transformation efficiency of competent cells should be review at different concentration.
Antibiotic concentration should be review.

10 June

Miniprep MerB and MerE (sample from DNA synthesis)

Procedure:
* Centrifuge E,coli transformed culture at 12000 rpm for 1 minute two times
* Resuspend in 250 µl resuspension buffer
* Add 250 µl of Lysis Buffer L7
* Add 350 µl of precipitation Buffer N4
* Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
* Centrifuge at 12000 rpm for a minute
* The supernatant was discarded and 500 µl of Wash Buffer were added
* The column was centrifuged at 12000 rpm for 1 minute
* 700 µl of Wash Buffer W9 with ethanol were added to the column
* The column in the washtube was centrifuged at 12000 rpm for 1 minute
* 75 µl of preheated TE Buffer were added
* The column was centrifuged at 12000 rpm for 2 minutes
Notes: Inventory season!, running gels must wait until next week

11 june

Miniprep PhyB, Pif6, NLS, VP16 and RFP 10 pg (Transformation efficiency)

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added
*The column was centrifuged at 12000 rpm for 2 minutes
Notes:Inventory season!, running gels must wait until next week

13 june

Media preparation (LB broth and LB agar)

Procedure
*14g of LB agar were weighted to prepare 400ml of medium
*1 g of LB broth were weighted to prepare 500 ml of medium
*Autoclave at 121°C for 15 minutes

17 june

Restriction digest of MerE, MerB and PSB1C3

Procedure:
*Add 10 ul of DNA to be digested, and 6 ul of dH20
*Add 2.5ul of NEBuffer 2.
*Add 0.5ul of BSA.
*Add 0.5ul of EcoRI.
*Add 0.5ul of PstI.
*Mix well and spin down briefly.
*Incubate the restriction digest at 37C for 30min, and then 80C for 20min
Notes:Mislabelling of sample, the experiment must be repeated

Ligation of MerE and MerB in PSB1C3

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample, 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
*Incubate 1 hour at 22°C. Inactivation of T4 DNA ligase at 65°C for 10 min
*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
Notes: T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants
Mislabelling of sample, the experiment must be repeated

18 june

Restriction digest of MerE, MerB and PSB1C3

Procedure:
*Add 10 ul of DNA to be digested, and 6 ul of dH20
*Add 2.5ul of NEBuffer 2.
*Add 0.5ul of BSA.
*Add 0.5ul of EcoRI.
*Add 0.5ul of PstI.
*Mix well and spin down briefly.
*Incubate the restriction digest at 37C for 30min, and then 80C for 20min

Ligation of MerE and MerB in PSB1C3

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample, 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
*Incubate 1 hour at 22°C.
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
Notes:T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants

Gel electrophoresis: PhyB, MerE, VP16, NLS, PIf6, MerB, RFP and PSB1C3


Procedure:
*2% agarose gel, the current was set to 100 V for 55 minutes. Results:
Notes:No band appears, there is not DNA sample in the gel. The transformation experiments must be repeated.

19 june

Gel Electrophoresis: MerB and MerE Restriction digest and ligation

Procedure:
*2% agarose gel, the current was set to 100 V for 55 minutes. Results:
Notes:No band appears, there is not DNA sample in the gel. The transformation, restriction digests and ligation experiments must be repeated.

20 june

Transformation of E.coli with MerB and MerE in PSB1C3

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Ampicillin (100mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes:To confirm that the gel has done correctly, parts ligated with PSB1C3 were transformed in E,coli After one week, no colonies growth in petri dishes

23 june

Competent cells

Procedure
*Add 1.5 ml of E.coli in LB broth to Ice tubes
*Centrifuged for 3 minutes at 6000 rpm, two times
*The supernatant was discarded
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
*Incubated in ice for 20 minutes
*The tubes were centrifuged again for 3 minutes at 6000 rpm
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c
Note: The first competent cells didn’t work as expected. The same protocol was applied this time.

Test 1: Transformation of E.coli with MerB, MerE, NLS, PhyB, VP16, AGS linker, PIF6, PolyA, TetR, RFP 10 pg (transformation efficiency)

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*2 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Chloramphenicol (35mg/ml) or Ampicillin(100mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes: The next day no colony appears. A second culture of the same SOC solution was done. The first colonies of MerB, MerE from the first culture appears two days later and the next day the second culture grew. But transformation efficiency kit and parts from the distribution kit are not working as expected.
Some colonies of Pif6 and RFP grew, after three days of incubation.
Antibiotic concentration of Chloramphenicol and competent cells efficiency should be review.

Terrific Broth preparation

Procedure:
*Weight 42.5 grams of Terrific Broth (Sigma-Aldrich) for each liter of media.
*Dilute in Erlenmeyer flasks with the adequate amount of distilled water.
*Autoclave

Pasar arabidopsis a tierra y medio

24 june

Inoculation of Transformed colonies of Test 1 in terrific broth (MerB, MerE, Pif6 and RFP)

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

Media preparation: LB agar with antibiotics at different concentrations

25 june

Competent cells

Procedure:
*Add 1.5 ml of E.coli in LB broth to Ice tubes
*Centrifuged for 3 minutes at 6000 rpm, two times
*The supernatant was discarded
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
*Incubated in ice for 20 minutes
*The tubes were centrifuged again for 3 minutes at 6000 rpm
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c
Notes: After the problems with test1, efficiency of competent cells it was not as expected. New competent cells was made.

Miniprep (1) MerB, MerE, RFP(te),Pif6

Aim of the experiment:
*Plasmid extraction from colonies of “transformation (test 1)” Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added
*The column was centrifuged at 12000 rpm for 2 minutes
Notes:

Test 2: Transformation of E.coli with MerB, MerE, NLS, PhyB, VP16, AGS linker, PIF6, PolyA, TetR, RFP(control)

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*2 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Chloramphenicol (15mg/ml) / Ampicillin (100 mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes:The colonies of MerB, MerE, NLS, AGS, Pif6, VP16 and TetR from the first culture appear the next day. But transformation efficiency kit and parts from the distribution kit are not working as expected. Antibiotic concentration of Chloramphenicol was changed to (15 mg/ml).

26 june

Miniprep (2) MerB, MerE, NLS, AGS, Pif6, VP16, TetR

Aim of the experiment:

Plasmid extraction from colonies of “transformation (test 2)”

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added
*The column was centrifuged at 12000 rpm for 2 minutes

27 june

Electrophoresis Gel of MerE, MerB, RFP


Results:

30 june

Electrophoresis gel of Miniprep: NLS, AGS, Pif6, VP16, TetR

1 july

Test1: Restriction digest TetR, Pif6, MerB, NLS, PolyA, MerE, MerB, PSB1C3

Procedure
***Note: some parts are A/B, cut them with EcoRI and PstI
*Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes
*Add 10 ul of DNA to be digested, and 6 ul of dH20
*Pipet 2.5ul of NEB Buffer 2 to each tube.
*Pipet 0.5ul of BSA to each tube.
*In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.
*In the Part B tube: Add 0.5ul of XbaI, and 0.5ul of PstI.
*In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
*Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture
*Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.

2 july

Test 1: Ligation

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample (4ul for sample A, 4 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
*Incubate 1 hour at 22°C.
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
Notes: T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants
Electrophoresis Gel (same samples as 30 july) and more samples

3 july

Electrophoresis gel: Restriction digests gel of Test 1

Electrophoresis gel: Ligation of test 1

4 july

Test 2: Restriction digest


***Note: for parts are A/B, cut with EcoRI and PstI
*Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes
*Add 10 ul of DNA to be digested, and 6 ul of dH20
*Pipet 2.5ul of NEB Buffer 2 to each tube.
*Pipet 0.5ul of BSA to each tube.
*In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.
*In the Part B tube: Add 0.5ul of XbaI, and 0.5ul of PstI.
*In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
*Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture
*Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.

7 july

Test 2: Ligation

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample (4ul for sample A, 4 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
*Incubate 1 hour at 22°C.
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
Notes: T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants

8 july

Electrophoresis gel: Restriction digest of test 2

Electrophoresis gel: Ligation of Test 2

9 july

Transformation of E.coli with Test 2

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes:

Rehydratation and Transformation PhyB


*Kit plates were rehydrated with 10 ul of distillated water
*50 µl of competent cells were placed in a cold eppendorf tube
*2 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

10 july

Test for antibiotic concentration

14 july

Restriction digests of linear plasmid PSB1A3

Procedure
*Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tube
*Add 10 ul of DNA to be digested, and 6 ul of dH20
*Pipet 2.5ul of NEB Buffer 2
*Pipet 0.5ul of BSA
*Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
*Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture
*Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.

Electrophoresis gel of PhyB

15 july

Competent cells

Procedure:
*Add 1.5 ml of E.coli in LB broth to Ice tubes
*Centrifuged for 3 minutes at 6000 rpm, two times
*The supernatant was discarded
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
*Incubated in ice for 20 minutes
*The tubes were centrifuged again for 3 minutes at 6000 rpm
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c

Ligation of Test

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample (4ul for sample A, 4 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
*Incubate 1 hour at 22°C.
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
Notes: T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants

16 july

Transformation of E.coli with

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes:

17 july

Last day of summer work!


Notes:Colonies for ligation never appear, all the minipreps were stored at -4°C to continue working on August!

18 august

Media preparation LB broth and LB agar and E.coli k.12, E.coli Top10 and agrobacterium inoculation

Procedure:
*14g of LB agar were weighted to prepare 400ml of medium
*Weight 42.5 grams of Terrific Broth for each liter of media.
*Autoclave
*E.coli was inoculated in petri dishes and incubated at 37°C
Notes:The growth mediums in storage were review; they didn’t show signs of contamination

19 august

Electrophoresis gel of summer minipreps


Notes:Most of the DNA from summer minipreps was loses. Probably, due to wrong storage conditions.

20 august

Transformation of E.coli with NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

21 august

Media preparation LB broth and E.coli Top10 inoculation

Procedure:
*14g of LB agar were weighted to prepare 400ml of medium
*1 g of LB broth were weighted to prepare 500 ml of medium
*Autoclave at 121°C for 15 minutes
*E.coli was inoculated in petri dishes and incubated at 37°C
Notes:The growth mediums in storage were review; they didn’t show signs of contamination

22 august

Calcium chloride competent cells

Procedure:
*Add 1.5 ml of E.coli in LB broth to Ice tubes
*Centrifuged for 3 minutes at 6000 rpm, two times
*The supernatant was discarded
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
*Incubated in ice for 20 minutes
*The tubes were centrifuged again for 3 minutes at 6000 rpm
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c

Transformation of E.coli with NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

25 august

Restricking plates with summer transformations in LB agar with antibiotics: NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS, MerE and MerB

26 august

Inoculation of Transformed colonies of summer transformations in terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

27 august

Miniprep: NLS, PhyB, VP16, PiF6, TetR, MerE, Pif6 and MerB

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel of minipreps: NLS, PhyB, VP16, PiF6, TetR, MerE, Pif6 and MerB

28 august

Transformation NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS, MerB and MerE

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

29 august

Inoculation of Transformed colonies of NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS, MerE and MerB in terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

1 september

Miniprep: MerB and MerE

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel miniprep: MerE y MerB

2 september

PCR

Electrophoresis Gel: PCR products

3 september

PCR

Electrophoresis Gel: PCR products

Transformation

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

4 september

Inoculation of Transformed colonies of PhyB, Pif6, AGS, NLS, VP16 and PhyB(2) in terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

5 september

Miniprep: PhyB, Pif6, AGS, NLS, VP16 and PhyB(2)

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel of Miniprep

8 september

E.coli top 10 inoculation

Procedure:

Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

Plasmid rehydratation of Agrobacterium vectors from paper filter.

9 september

E.coli top10 Electrocompent cells

Procedure:
*Dilute the cells 100X. 100 ml LB Amp. Grow at OD600 between 0.6 and 0.8.
*Split the culture into 2x 50 ml falcon tubes, on ice.
*Centrifuge at 4 °C for 10 min at 4000 rpm.
*Wash and combine the cells. Remove the supernatant.
*Resuspend the cells in 2x 25 ml of ice cold water. Combine the volumes
*Wash the cells 2 more times and entrifuge at 4 °C for 10 min at 4000 rpm.
*Resuspend in 50 ml of ice cold water and Repeat.
*Wash and concentrate the cells and Centrifuge at 4 °C for 10 min at 4000 rpm.
*Resuspend in 1-2 ml of ice cold water and storage at -20°C

10 september

Electro-Transformation of RBS, VP16, Pif6, Pif6 (2) and TetR

Procedure:
*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
*******Electroporation:
*******Mode Prokaryotes
*******Voltage (V) 1,700 V
*******Time constant (τ) 5 ms
*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
*Plate on LB plates with Ampicillin.

11 september

Inoculation of E.coli with RBS, VP16, Pif6, Pif6 (2) and TetR in Terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

12 september

Miniprep: RBS, VP16, Pif6, Pif6 (2) and TetR

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis Gel of miniprep [RBS, VP16, Pif6, Pif6 (2) and TetR]

13 september

Restriction digest of MerB, MerE and Psc1c3

Procedure:
*Add 9 ul of DNA to be digested, and 6 ul of dH20
*Pipet 2.5ul of NEB Buffer 2 to each tube.
*Pipet 0.5ul of BSA to each tube.
*In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of PstI.
*In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI,
*Incubate the restriction digests at 37°C for 60 minutes

Electrophoresis gel: Restriction digest of MerB, MerE

14 september

Restriction digest of MerB, MerE and Psc1c3 [NEW ENZYMES]

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min
Notes:

Electrophoresis gel: Restriction digest of MerB, MerE

DNA Gel extraction

Procedure
*Place 400 mg of the excised gel into a 1.5-mL tube.
*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
*Place the tube into a 50°C water bath and at least 10 minutes.
*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
*Store the purified DNA at 4°C

15 september

Nanodrop

Ligation of MerB and MerE in Psb1c3

E. coli Electrocompetent cells

Procedure:
*Dilute the cells 100X. 100 ml LB Amp. Grow at OD600 between 0.6 and 0.8.
*Split the culture into 2x 50 ml falcon tubes, on ice.
*Centrifuge at 4 °C for 10 min at 4000 rpm.
*Wash and combine the cells. Remove the supernatant.
*Resuspend the cells in 2x 25 ml of ice cold water. Combine the volumes
*Wash the cells 2 more times and entrifuge at 4 °C for 10 min at 4000 rpm.
*Resuspend in 50 ml of ice cold water and Repeat.
*Wash and concentrate the cells and Centrifuge at 4 °C for 10 min at 4000 rpm.
*Resuspend in 1-2 ml of ice cold water and storage at -20°C

16 september

Electrotransformation of MerB and MerE in Psb1c3

Procedure:
*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
*******Electroporation:
*******Mode Prokaryotes
*******Voltage (V) 1,700 V
*******Time constant (τ) 5 ms
*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
*Plate on LB plates with Ampicillin.

17 september

Electrotransformation of MerB and MerE in Psb1c3

Procedure:
*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
*******Electroporation:
*******Mode Prokaryotes
*******Voltage (V) 1,700 V
*******Time constant (τ) 5 ms
*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
*Plate on LB plates with Chloramphenicol.

18 september

Inoculation in Terrific broth [MerB and MerE in Psb1c3]

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

19 september

Miniprep: MerB and MerE in Psb1c3

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel [Miniprep: MerB and MerE in Psb1c3]

20 september

Creating tools:

Media preparation LB broth and LB agar

Procedure:
*14g of LB agar were weighted to prepare 400ml of medium
*1 g of LB broth were weighted to prepare 500 ml of medium
*Autoclave at 121°C for 15 minutes
*E.coli was inoculated in petri dishes and incubated at 37°C

Stocks: Ampicillin, Kanamycin and Chloramphenicol

Procedure:
*******Chloramphenicol Stock:
*Weight 10 mg of lyophilized antibiotic for each ml of stock solution.
*Dissolve in ethanol and mix gently (It is possible to use Vortex)
*******Kanamicin Stock:
*Weight 50 mg of lyophilized antibiotic for each ml of stock solution.
*Dissolve in distilled water and mix gently (It is possible to use Vortex).
*******Ampicilin Stock:
*Weight 100 mg of liophylized antibiotic for each ml of stock solution.
*Dissolve in distilled water and mix gently (It is possible to use Vortex).
*******Working concentration for each antibiotic will be:
*Cloramphenicol: 5l/ml
*Kanamicin: 25 l/ml
*Ampicilin: 50 l/ml

Inoculation E.coli in Terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

21 september

E. coli Calcium Chloride competent cell protocol


*Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning and shake @ 37°C for 3hrs.
*Put the cells on ice for 10 min and collect the cells by centrifugation for 3 mins @6krpm
*Decant supernatant and gently resuspend on 10 mL cold 0.1M CaCl
*Incubate on ice x 20 mins and centrifuge 3 mins @6krpm
*Discard supernatant and gently resuspend on 5mL cold 0.1MCaCl/15%Glycerol
*Dispense in microtubes (300μL/tube). Freeze in -80°C.

Transformation: Measure competent cells Transformation efficiency with PGLO

22 september

Restriction digest RBS(A), PhyB(B), AGS(A), VP16(B), MerE(C), MerB(C), NLS (A) and PolyA(B)

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min

Electrophoresis gel of restriction digest: RBS, PhyB, AGS, VP16, MerE, MerB, NLS and PolyA

DNA Gel extraction

Procedure
*Place 400 mg of the excised gel into a 1.5-mL tube.
*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
*Place the tube into a 50°C water bath and at least 10 minutes.
*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
*Store the purified DNA at 4°C

23 september

Ligation RBS with PhyB, AGS with VP16, RBS with MerE, RBS with MerB and NLS with PolyA

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 12 ul of DNA sample (6ul for sample A, 6 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of nuclease free water.
*Incubate 1 hour at 22°C. (termocycle)
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use 10 μL of the mixture for transformation of 50 μL of chemically competent cells

Electrotransformation of E.coli with [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA]

Procedure:
*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
********Electroporation:
********Mode Prokaryotes
********Voltage (V) 1,700 V
********Time constant (τ) 5 ms
*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
*Plate on LB plates.

24 september

Inoculation of E.coli with [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA] in Terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

25 september

Miniprep [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA]

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel of Miniprep: [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA]

26 september

Restriction digest [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and V3]

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min

Electrophoresis Gel of restriction digest: [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and V3]

DNA Gel extraction

Procedure
*Place 400 mg of the excised gel into a 1.5-mL tube.
*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
*Place the tube into a 50°C water bath and at least 10 minutes.
*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
*Store the purified DNA at 4°C Ligation of RBS-MerE with RBS-MerB, RBS-PhyB with AGS-VP16 and of RBS-MerE with RBS-MerB in V3. Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 12 ul of DNA sample (6ul for sample A, 6 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of nuclease free water.
*Incubate 1 hour at 22°C. (termocycle)
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use 10 μL of the mixture for transformation of 50 μL of chemically competent cells

Transformation of E.coli and Agrobacterium with [RBS-MerE-RBS-MerB and RBS-PhyB-AGS-VP16]

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*10 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 60 seconds
*The tube was placed in ice for 5 minutes
*200 µl of S.O.C. were added
*The tube was left incubating for 2 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

27 september

Inoculation of E.coli and Agrobacterium with [RBS-MerE-RBS-MerB] in Terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

28 september

Miniprep [RBS-MerE-RBS-MerB]

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel of Miniprep: [RBS-MerE-RBS-MerB]

29 september

Restriction digest of RBS-PhyB and AGS-VP16

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min

Electrophoresis Gel of Restriction digest [RBS-PhyB and AGS-VP16]

30 september

Restriction digest of RBS-PhyB and AGS-VP16

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min

Electrophoresis Gel of Restriction digest [RBS-PhyB and AGS-VP16]

DNA gel extraction

Procedure
*Place 400 mg of the excised gel into a 1.5-mL tube.
*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
*Place the tube into a 50°C water bath and at least 10 minutes.
*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
*Store the purified DNA at 4°C

1 october

Ligation of RBS-PhyB with AGS-VP16

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 12 ul of DNA sample (6ul for sample A, 6 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of nuclease free water.
*Incubate 1 hour at 22°C. (termocycle)
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use 10 μL of the mixture for transformation of 50 μL of chemically competent cells

Transformation of E.coli with [RBS-PhyB-AGS-VP16]

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*10 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 60 seconds
*The tube was placed in ice for 5 minutes
*200 µl of S.O.C. were added
*The tube was left incubating for 2 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

2 october

Inoculation of E.coli with [RBS-PhyB-AGS-VP16] in Terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

3 october

Miniprep [RBS-PhyB-AGS-VP16]

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel of Miniprep: [RBS-PhyB-AGS-VP16]

4 october

Restriction digest of RBS-PhyB-AGS-VP16 and NLS-PolyA

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min
Notes:

Electrophoresis gel of Restriction digest [RBS-PhyB-AGS-VP16 and NLS-PolyA]

DNA Gel purification

Procedure
*Place 400 mg of the excised gel into a 1.5-mL tube.
*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
*Place the tube into a 50°C water bath and at least 10 minutes.
*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
*Store the purified DNA at 4°C

Ligation of RBS-PhyB-AGS-VP16 with NLS-PolyA

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 12 ul of DNA sample (6ul for sample A, 6 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of nuclease free water.
*Incubate 1 hour at 22°C. (termocycle)
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use 10 μL of the mixture for transformation of 50 μL of chemically competent cells

5 october

Transformation of E.coli with [RBS-PhyB-AGS-VP16-NLS-PolyA]

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*10 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 60 seconds
*The tube was placed in ice for 5 minutes
*200 µl of S.O.C. were added
*The tube was left incubating for 2 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

6 october

Inoculation of E.coli with [RBS-PhyB-AGS-VP16-NLS-PolyA] in Terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

7 october

Miniprep [RBS-PhyB-AGS-VP16-NLS-PolyA]

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel of Miniprep: [RBS-PhyB-AGS-VP16-NLS-PolyA]

8 october

Retrieved from "http://2014.igem.org/Team:BIOSINT_Mexico/lab_records"