Team:Marburg:Project:Notebook:Methods

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Line 12: Line 12:
<div class="method">
<div class="method">
<fieldset class="competent_cells">
<fieldset class="competent_cells">
-
     <legend><a name="competent_cells">Competent Cells</a></legend>
+
     <legend><a name="competent_cells">Competent Cells <i>Escherichia coli</i></a></legend>
     <div class="exp-content">
     <div class="exp-content">
     <p><strong>1. Peparation for preculture</strong></p>
     <p><strong>1. Peparation for preculture</strong></p>
<ul class="comp">
<ul class="comp">
<li>50 &micro;L Aliquod in 2x5ml LB  each</li>
<li>50 &micro;L Aliquod in 2x5ml LB  each</li>
-
<li>incubation Overnight at 37&deg;C   </li>
+
<li>incubation Overnight at 37&deg;C  </li>
         </ul>
         </ul>
       <p><strong>2. Preparation for main culture</strong></p>
       <p><strong>2. Preparation for main culture</strong></p>
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     <p><strong>1. Transformation</strong></p>
     <p><strong>1. Transformation</strong></p>
<ul class="comp">
<ul class="comp">
-
  <li>transfer 1 µL plasmid into aliquots for 10min on ice</li>
+
  <li>transfer 1 Â&micro;L plasmid into aliquots for 10min on ice</li>
-
<li>45sec. Heat shock at 42°C</li>
+
<li>45sec. Heat shock at 42°C</li>
<li>10 min on ice</li>
<li>10 min on ice</li>
       </ul>
       </ul>
       <p><strong>2. Cultivation of transformed cells</strong></p>
       <p><strong>2. Cultivation of transformed cells</strong></p>
         <ul class="comp">
         <ul class="comp">
-
<li>add 700 µL LB to transformed cells                                                                                                                                                                     </li>
+
<li>add 700 Â&micro;L LB to transformed cells                                                                                                                                                                     </li>
-
<li>incubation for 1,5h at 37°C</li>
+
<li>incubation for 1,5h at 37°C</li>
<li>centrifugation for 3min at 13000rpm</li>
<li>centrifugation for 3min at 13000rpm</li>
-
<li>get off 600 µL per 1,5ml tube</li>
+
<li>get off 600 Â&micro;L per 1,5ml tube</li>
-
<li>resuspend pellet in left 100µL LB</li>
+
<li>resuspend pellet in left 100Â&micro;L LB</li>
<li>plating on agar plate</li>
<li>plating on agar plate</li>
<li>incubation overnight</li>
<li>incubation overnight</li>
Line 74: Line 74:
     <p><strong>1. Picking colonies</strong></p>
     <p><strong>1. Picking colonies</strong></p>
<ul class="comp">
<ul class="comp">
-
  <li>inoculate 20 ml LB medium (sterile) containing 20µL antibiotics**  and incubate until  an OD of 0,6</li>
+
  <li>inoculate 20 ml LB medium (sterile) containing 20Â&micro;L antibiotics** and incubate until  an OD of 0,6</li>
-
<li>45sec. Heat shock at 42°C</li>
+
<li>45sec. Heat shock at 42°C</li>
<li>10 min on ice</li>
<li>10 min on ice</li>
       </ul>
       </ul>
       <p><strong>2. Induction with IPTG/ Lactose</strong></p>
       <p><strong>2. Induction with IPTG/ Lactose</strong></p>
         <ul class="comp">
         <ul class="comp">
-
  <li>before induction:  0,7/ OD = sample  volume</li>
+
  <li>before induction: 0,7/ OD = sample  volume</li>
-
<li>pellet + 80 µL water were resuspended in 20µL loading buffer  (preinduction sample) </li>
+
<li>pellet + 80 Â&micro;L water were resuspended in 20Â&micro;L loading buffer  (preinduction sample) </li>
-
<li>induction with 100 µL IPTG/ lactose at an OD of 0,6</li>
+
<li>induction with 100 Â&micro;L IPTG/ lactose at an OD of 0,6</li>
-
<li>incubation for 1,5h    - 4 h max</li>
+
<li>incubation for 1,5h    - 4 h max</li>
-
<li>after induction:  200 µL sample    </li>
+
<li>after induction: 200 Â&micro;L sample    </li>
-
<li>pellet  +80 µL water + 20 µL loading buffer  resuspended (induction sample)</li>
+
<li>pellet  +80 Â&micro;L water + 20 Â&micro;L loading buffer  resuspended (induction sample)</li>
-
<li>big volume gap because of   decreased growth of bacteria   <br />
+
<li>big volume gap because of  decreased growth of bacteria  <br />
    after induction</li>
    after induction</li>
       </ul>
       </ul>
Line 92: Line 92:
         <ul class="comp">
         <ul class="comp">
  <li>transfer in 2x50ml falcons</li>
  <li>transfer in 2x50ml falcons</li>
-
<li>centrifugation at 4°C, 3500rpm, 15min</li>
+
<li>centrifugation at 4°C, 3500rpm, 15min</li>
<li>washing  pellets in 10ml buffer A</li>
<li>washing  pellets in 10ml buffer A</li>
<li>cracking  cells with micro fluidizer</li>
<li>cracking  cells with micro fluidizer</li>
-
         <li>thermo  centrifugation for 20 min – 4°C</li>
+
         <li>thermo  centrifugation for 20 min – 4°C</li>
-
    <li>200 µL Ni-NTA beats +  supernatant</li>
+
    <li>200 Â&micro;L Ni-NTA beats +  supernatant</li>
           <li>5min 4000  rpm &rarr; pellet</li>
           <li>5min 4000  rpm &rarr; pellet</li>
-
           <li>resuspended  pellet in 500 µL Buffer A (low imidazole lv)</li>
+
           <li>resuspended  pellet in 500 Â&micro;L Buffer A (low imidazole lv)</li>
-
           <li>centrifugation  1 min – 4000rpm</li>
+
           <li>centrifugation  1 min – 4000rpm</li>
           <li>pellet  resuspended in buffer A</li>
           <li>pellet  resuspended in buffer A</li>
-
           <li>centrifugation  1 min – 4000rpm</li>
+
           <li>centrifugation  1 min – 4000rpm</li>
-
           <li>pellet  resuspended in 200 µL Buffer B (elution)</li>
+
           <li>pellet  resuspended in 200 Â&micro;L Buffer B (elution)</li>
-
           <li>centrifugation  1min – 13000</li>
+
           <li>centrifugation  1min – 13000</li>
-
           <li>80µL supernatant (incl.  protein)+ 20µL loading buffer</li>
+
           <li>80Â&micro;L supernatant (incl.  protein)+ 20Â&micro;L loading buffer</li>
       </ul>
       </ul>
           <p><strong>4. Expression test with induced culture &rarr; SDS-Gel</strong></p>
           <p><strong>4. Expression test with induced culture &rarr; SDS-Gel</strong></p>
<ul class="comp">
<ul class="comp">
-
    <li>ladder – 5 µL</li>
+
    <li>ladder – 5 Â&micro;L</li>
-
<li>PI– preinduction sample - 10µL</li>
+
<li>PI– preinduction sample - 10Â&micro;L</li>
-
<li>I    - induction sample -10 µL</li>
+
<li>I    - induction sample -10 Â&micro;L</li>
-
<li>E    -elution sample -10 µL</li>
+
<li>E    -elution sample -10 Â&micro;L</li>
       </ul>
       </ul>
</div>
</div>
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<div class="method">
<div class="method">
<fieldset class="comp_bac">
<fieldset class="comp_bac">
-
     <legend><a name="comp_bac">Making Competent Bacillus</a></legend>
+
     <legend><a name="comp_bac">Making Competent <i>Bacillus subtilis</i></a></legend>
     <div class="exp-content">
     <div class="exp-content">
-
     <p>B. subtilis (PY79) is naturally competent (Albano et al., 1987). In order to increase the transformation rate, Bacillus can be grown in special media to enhance the competence. To achieve this, 20ml SPC-medium was inoculated with half a well grown LB-plate and incubated at 37°C until the cells reached the stationary phase. (Optical density OD550 nm does not change within a a timespan of 30 min) The cells were transfered into 100ml SPII-medium and incubated for 90min at 37°C. Afterwards, the cells were centrifuged (500 rpm, 15min, Rotor: BS4402/A Heraeus). The pellet was resuspended in 1ml glycerine (50% (v/v)). Samples were aliquoted and stored at -80°C.</p>
+
     <p>B. subtilis (PY79) is naturally competent (Albano et al., 1987). In order to increase the transformation rate, Bacillus can be grown in special media to enhance the competence. To achieve this, 20ml SPC-medium was inoculated with half a well grown LB-plate and incubated at 37°C until the cells reached the stationary phase. (Optical density OD550 nm does not change within a a timespan of 30 min) The cells were transfered into 100ml SPII-medium and incubated for 90min at 37°C. Afterwards, the cells were centrifuged (500 rpm, 15min, Rotor: BS4402/A Heraeus). The pellet was resuspended in 1ml glycerine (50% (v/v)). Samples were aliquoted and stored at -80°C.</p>
-
     <p>For the transformation with plasmid-DNA 100µl cells and 5-7µl DNA (chromosomal DNA: 0,1-1µl DNA) were mixed andd incubated at 37°C for 30min. Afterwards, LB-plates (with antibiotics for selection) were inoculated with the culture and incubated at 30°C until colonies formed.</p>
+
     <p>For the transformation with plasmid-DNA 100Â&micro;l cells and 5-7Â&micro;l DNA (chromosomal DNA: 0,1-1Â&micro;l DNA) were mixed andd incubated at 37°C for 30min. Afterwards, LB-plates (with antibiotics for selection) were inoculated with the culture and incubated at 30°C until colonies formed.</p>
     <p><strong>SPC-Medium: </strong></p>
     <p><strong>SPC-Medium: </strong></p>
     <table width="100%" border="1">
     <table width="100%" border="1">
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         <td>CaCl2 </td>
         <td>CaCl2 </td>
         <td>0,1 M </td>
         <td>0,1 M </td>
-
         <td>500 μl </td>
+
         <td>500 μl </td>
       </tr>
       </tr>
       <tr>
       <tr>
Line 235: Line 235:
           <li>add 700&micro;l DNA Wash Buffer per tube</li>
           <li>add 700&micro;l DNA Wash Buffer per tube</li>
           <li>centrifugation for 1min throw filrate away</li>
           <li>centrifugation for 1min throw filrate away</li>
-
           <li>add 30&micro;l milipore per tube – let column sit for 2min</li>
+
           <li>add 30&micro;l milipore per tube – let column sit for 2min</li>
           <li>centrifuge for 2min</li>
           <li>centrifuge for 2min</li>
</ul>
</ul>
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<li>weigh the gel with the tube</li>
<li>weigh the gel with the tube</li>
<li>add QG (100mg gel &rarr; 100&micro;l)</li>
<li>add QG (100mg gel &rarr; 100&micro;l)</li>
-
<li>incubate for approx. 10min at 50-60°C, vortex to dissolve the gel</li>
+
<li>incubate for approx. 10min at 50-60°C, vortex to dissolve the gel</li>
             <li>put spin colum into 2mlcollection tube</li>
             <li>put spin colum into 2mlcollection tube</li>
             <li>add dissolved gel onto column &rarr; centrifuge for 1min</li>
             <li>add dissolved gel onto column &rarr; centrifuge for 1min</li>
             <li>discard flow-through</li>
             <li>discard flow-through</li>
-
             <li>wash with 750µl Buffer PE &rarr;
+
             <li>wash with 750Â&micro;l Buffer PE &rarr;
centrifuge for 1min          <li>discard flow-through</li>
centrifuge for 1min          <li>discard flow-through</li>
             <li>place column in 1,5ml tube</li>
             <li>place column in 1,5ml tube</li>
-
             <li>elute with 50µl milipore water: incubate 5min at 37°C &rarr;
+
             <li>elute with 50Â&micro;l milipore water: incubate 5min at 37°C &rarr;
centrifuge for 1min</li>
centrifuge for 1min</li>
</ul>
</ul>
Line 274: Line 274:
     <p><strong>1. Preculture for competent Bacillus subtilis</strong></p>
     <p><strong>1. Preculture for competent Bacillus subtilis</strong></p>
     <p>Aim:Preparation for mainculture the next day</p>
     <p>Aim:Preparation for mainculture the next day</p>
-
     <p> 5 mL of LB were inoculated with Bacillus WT3610 from an LB plate and  incubated at 37°C overnight.</p>
+
     <p> 5 mL of LB were inoculated with Bacillus WT3610 from an LB plate and  incubated at 37°C overnight.</p>
<p><strong>2. Transformation of competent Bacillus subtilis</strong></p>
<p><strong>2. Transformation of competent Bacillus subtilis</strong></p>
<p>Aim: transformation of plasmid into Bacillus subtilis WT3610</p>
<p>Aim: transformation of plasmid into Bacillus subtilis WT3610</p>
-
<p>100 µL of overnight culture were added to 10  mL of MNGE-Medium and incubated till an OD of 1,1-1,3 at 37°C which could take  4-5h.4</p>
+
<p>100 Â&micro;L of overnight culture were added to 10  mL of MNGE-Medium and incubated till an OD of 1,1-1,3 at 37°C which could take  4-5h.4</p>
-
<p>After reaching OD of 1,1-1,3 400 µL of the  culture were transformed with 1,5 µg plasmid. After 1h incubation at 37°C 100  µL Expression mix were added and incubated for 1h as well.</p>
+
<p>After reaching OD of 1,1-1,3 400 Â&micro;L of the  culture were transformed with 1,5 Â&micro;g plasmid. After 1h incubation at 37°C 100  Â&micro;L Expression mix were added and incubated for 1h as well.</p>
-
<p>In the end the 500 µL attempt was plated out  on MLS-X-Gal plates and incubated at 30 °C overnight until colonies could be  seen. </p>
+
<p>In the end the 500 Â&micro;L attempt was plated out  on MLS-X-Gal plates and incubated at 30 °C overnight until colonies could be  seen. </p>
<p><strong>3. Overnight culture of blue clones</strong></p>
<p><strong>3. Overnight culture of blue clones</strong></p>
<p>Aim: transformation of plasmid into Bacillus subtilis WT3610</p>
<p>Aim: transformation of plasmid into Bacillus subtilis WT3610</p>
-
     <p>Colonies were grown on the plates with  transformed plasmid. The blue/ white screening showed positive transformed blue  clones.  3 clones of different morphology  per plate were picked and used for inoculation of LB-MLS (4 mL LB, 4 µL Lincomycin, 4 µL Erythromycin). Incubation was carried out overnight at 30°C with the cultures. </p>
+
     <p>Colonies were grown on the plates with  transformed plasmid. The blue/ white screening showed positive transformed blue  clones. 3 clones of different morphology  per plate were picked and used for inoculation of LB-MLS (4 mL LB, 4 Â&micro;L Lincomycin, 4 Â&micro;L Erythromycin). Incubation was carried out overnight at 30°C with the cultures. </p>
     <p><strong>4. First temperature shift</strong></p>
     <p><strong>4. First temperature shift</strong></p>
-
     <p>Aim: integration of  pMAD-Insert  into Bacillus chromosome via flanks</p>
+
     <p>Aim: integration of  pMAD-Insert  into Bacillus chromosome via flanks</p>
-
     <p>The   overnight cultures were used to inoculate 10 mL LB MLS until  the culture obtained an OD of 0,1. The  cultures were incubated at 30°C for 2h.</p>
+
     <p>The  overnight cultures were used to inoculate 10 mL LB MLS until  the culture obtained an OD of 0,1. The  cultures were incubated at 30°C for 2h.</p>
-
     <p>Then the temperature was shifted to 42°C for  6h.</p>
+
     <p>Then the temperature was shifted to 42°C for  6h.</p>
-
     <p>After the heat shock dilutions from 10-4  to 10-6 of each culture were plated out on MLS-X-Gal so that plates  could be incubated overnight at 42°C.</p>
+
     <p>After the heat shock dilutions from 10-4  to 10-6 of each culture were plated out on MLS-X-Gal so that plates  could be incubated overnight at 42°C.</p>
     <p><strong>5. Second temperature shift</strong></p>
     <p><strong>5. Second temperature shift</strong></p>
     <p>Aim: flip out of the pMAD backbone</p>
     <p>Aim: flip out of the pMAD backbone</p>
-
     <p>One blue colony per diluted clone was used to  inoculate 4 mL LB. The cultures were incubated at 30°C for 6h and afterwards  for 3h at 42°C.</p>
+
     <p>One blue colony per diluted clone was used to  inoculate 4 mL LB. The cultures were incubated at 30°C for 6h and afterwards  for 3h at 42°C.</p>
-
     <p>Dilutions from 10-4­ to 10-6  were plated out on  X-Gal plates WITHOUT  MLS selection. The positive clones should not contain the resistance inside the  backbone as well as the galactosidase. The plates were incubated at 42°C overnight.</p>
+
     <p>Dilutions from 10-4­ to 10-6  were plated out on  X-Gal plates WITHOUT  MLS selection. The positive clones should not contain the resistance inside the  backbone as well as the galactosidase. The plates were incubated at 42°C overnight.</p>
     <p><strong>6.1 selection of positive clones</strong></p>
     <p><strong>6.1 selection of positive clones</strong></p>
     <p>Aim: checking the correct flip out of the pMAD backbone</p>
     <p>Aim: checking the correct flip out of the pMAD backbone</p>
     <p>From the dilution plates was one WHITE clone  picked and transferred on a Master X-Gal Plate as well as on a MLS plate so  that clones were proven for the right integration of the insert although  flipping out the pMAD backbone.</p>
     <p>From the dilution plates was one WHITE clone  picked and transferred on a Master X-Gal Plate as well as on a MLS plate so  that clones were proven for the right integration of the insert although  flipping out the pMAD backbone.</p>
-
     <p>The plates were incubated at 42°C overnight.</p>
+
     <p>The plates were incubated at 42°C overnight.</p>
     <p><strong>6.2 selection of positive clones</strong></p>
     <p><strong>6.2 selection of positive clones</strong></p>
     <p>Aim: checking the correct flip out of the pMAD backbone</p>
     <p>Aim: checking the correct flip out of the pMAD backbone</p>
Line 301: Line 301:
     <p><strong>7. cPCR with checked clones</strong></p>
     <p><strong>7. cPCR with checked clones</strong></p>
     <p>Aim: checking integration of constructs Hag-Spe and Hag-Spe-DARPin into  B. s. genome</p>
     <p>Aim: checking integration of constructs Hag-Spe and Hag-Spe-DARPin into  B. s. genome</p>
-
     <p>No clone was growing on MLS plates so the  picked clones seemed to be positive. In order to check the correct integration  of the domain constructs a cPCR with the picked clones on the X-Gal plates was  done. The picked B.s. clones were cooked in 10 µL PBS for 5 min at 95°C.</p>
+
     <p>No clone was growing on MLS plates so the  picked clones seemed to be positive. In order to check the correct integration  of the domain constructs a cPCR with the picked clones on the X-Gal plates was  done. The picked B.s. clones were cooked in 10 Â&micro;L PBS for 5 min at 95°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 313: Line 313:
     <div class="exp-content">
     <div class="exp-content">
     <p><em>modified after Harwood and Cutting 1990</em></p>
     <p><em>modified after Harwood and Cutting 1990</em></p>
-
     <p>LB-plates were inoculated with Bacillus subtilies and incubated at 37°C overnight.</p>
+
     <p>LB-plates were inoculated with Bacillus subtilies and incubated at 37°C overnight.</p>
-
     <p>3ml HS- (high-salt-) medium were inoculated with a single colony and incubated at 37°C overnight (rolling).</p>
+
     <p>3ml HS- (high-salt-) medium were inoculated with a single colony and incubated at 37°C overnight (rolling).</p>
-
     <p>20ml preheated LS- (low salt-) medium was inoculated with 1ml HS-culture and incubated at 30°C for 3h at 100rpm in a water bath.</p>
+
     <p>20ml preheated LS- (low salt-) medium was inoculated with 1ml HS-culture and incubated at 30°C for 3h at 100rpm in a water bath.</p>
     <p><strong>10 x S-Base</strong></p>
     <p><strong>10 x S-Base</strong></p>
     <table width="100%" border="1">
     <table width="100%" border="1">
Line 398: Line 398:
   <tr>
   <tr>
     <td>Tryptophane 5 mg/ml </td>
     <td>Tryptophane 5 mg/ml </td>
-
     <td>20 µl</td>
+
     <td>20 Â&micro;l</td>
   </tr>
   </tr>
   <tr>
   <tr>
     <td>Phenylalanine 3 mg/ml </td>
     <td>Phenylalanine 3 mg/ml </td>
-
     <td>30 µl</td>
+
     <td>30 Â&micro;l</td>
   </tr>
   </tr>
   <tr>
   <tr>
Line 418: Line 418:
   <tr>
   <tr>
     <td>MgCl2 1M</td>
     <td>MgCl2 1M</td>
-
     <td>50 µl</td>
+
     <td>50 Â&micro;l</td>
   </tr>
   </tr>
   <tr>
   <tr>
Line 426: Line 426:
     </table>
     </table>
     <p><strong>Transformation</strong></p>
     <p><strong>Transformation</strong></p>
-
     <p>Prepare DNA-Solution (1-5µg plasmid DNA or 10-20µg chromosomal DNA) in eppendorf cup.<br />
+
     <p>Prepare DNA-Solution (1-5Â&micro;g plasmid DNA or 10-20Â&micro;g chromosomal DNA) in eppendorf cup.<br />
-
       Add 1ml LS-culture, incubate the transformation samples at 37°C for 2h an a thermomixer/roller.<br />
+
       Add 1ml LS-culture, incubate the transformation samples at 37°C for 2h an a thermomixer/roller.<br />
       Centrifuge the transformation samples fpr 20sec, discard some of the supernatant and resuspend the cells.<br />
       Centrifuge the transformation samples fpr 20sec, discard some of the supernatant and resuspend the cells.<br />
       Incubate selective plates.</p>
       Incubate selective plates.</p>
Line 442: Line 442:
     <div class="exp-content">
     <div class="exp-content">
     <ul>
     <ul>
-
       <li>10 ml LB  inoculated with single colony as preculture over night at 37°C</li>
+
       <li>10 ml LB  inoculated with single colony as preculture over night at 37°C</li>
       <li>Inoculate  10 ml Spizizens minimal medium with the preculture to an OD600 of  0.1</li>
       <li>Inoculate  10 ml Spizizens minimal medium with the preculture to an OD600 of  0.1</li>
-
       <li>Incubation  at 37°C until OD600 1 more or less</li>
+
       <li>Incubation  at 37°C until OD600 1 more or less</li>
-
       <li>Addition  of 5 µg plasmid DNA/5-10 µg of chromosomal DNA to 1 ml of culture (in a test  tube)</li>
+
       <li>Addition  of 5 Â&micro;g plasmid DNA/5-10 Â&micro;g of chromosomal DNA to 1 ml of culture (in a test  tube)</li>
-
       <li>Incubation  at 37°C for 2 h</li>
+
       <li>Incubation  at 37°C for 2 h</li>
-
<li>Plate out 200 µl and 500 µl on different plates containing selecting  antibiotics and incubate the plates at 37°C overnight</li>
+
<li>Plate out 200 Â&micro;l and 500 Â&micro;l on different plates containing selecting  antibiotics and incubate the plates at 37°C overnight</li>
     </ul>
     </ul>
</div>
</div>
Line 462: Line 462:
     <ul class="list">
     <ul class="list">
     <li>equilibration with Buffer A 10 min</li>
     <li>equilibration with Buffer A 10 min</li>
-
     <li>taking 40 µL supernatant  (load)+  10 µL SDS-Buffer - L-sample</li>
+
     <li>taking 40 Â&micro;L supernatant  (load)+  10 Â&micro;L SDS-Buffer - L-sample</li>
     <li>50 mL load on column</li>
     <li>50 mL load on column</li>
-
     <li>taking 40 µL of flow through + 10 µL SDS-buffer - FT-sample</li>
+
     <li>taking 40 Â&micro;L of flow through + 10 Â&micro;L SDS-buffer - FT-sample</li>
     <li>first washing with 25ml Buffer A (half the  load)</li>
     <li>first washing with 25ml Buffer A (half the  load)</li>
-
     <li>taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-sample</li>
+
     <li>taking 40 Â&micro;L of washing flow through + 10 Â&micro;L SDS-Buffer - W-sample</li>
     <li>equilibrate with Buffer B (output pipe off the column into glas, input  into B)</li>
     <li>equilibrate with Buffer B (output pipe off the column into glas, input  into B)</li>
     <li>hanging pipe on column, 20 ml Elution - E</li>
     <li>hanging pipe on column, 20 ml Elution - E</li>
-
     <li>taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E- sample </li>
+
     <li>taking 40 Â&micro;L of Elution 1-6 + 10 Â&micro;L SDS-Buffer E- sample </li>
     <li>SDS-PAGE analysis</li>
     <li>SDS-PAGE analysis</li>
     </ul>
     </ul>
Line 490: Line 490:
     <legend><a name="split_caco2">Splitting of Caco-2-cells</a></legend>
     <legend><a name="split_caco2">Splitting of Caco-2-cells</a></legend>
     <div class="exp-content">
     <div class="exp-content">
-
     <p>Caco-2-cells require  DMEM (Dulbecco’s Modified Eagle Medium) with 20% FCS and L-Glutamine.</p>
+
     <p>Caco-2-cells require  DMEM (Dulbecco’s Modified Eagle Medium) with 20% FCS and L-Glutamine.</p>
-
     <p>A centrifuge was set  on 4°C.</p>
+
     <p>A centrifuge was set  on 4°C.</p>
     <p>The medium of the  culture flask was discarded from the side opposite to the adherent cells and  washed with 1X PBS from the opposite site as well.</p>
     <p>The medium of the  culture flask was discarded from the side opposite to the adherent cells and  washed with 1X PBS from the opposite site as well.</p>
-
     <p>The PBS was aspirated  with a glass pipette until the supernatant was clear. After adding 1-2 mL 1x  trypsin from the opposite of the adherent cells the flask was incubated for  5-10 min at 37°C depending on how fast the cells come of from the ground. Under  the microscope the free floating cells were checked.</p>
+
     <p>The PBS was aspirated  with a glass pipette until the supernatant was clear. After adding 1-2 mL 1x  trypsin from the opposite of the adherent cells the flask was incubated for  5-10 min at 37°C depending on how fast the cells come of from the ground. Under  the microscope the free floating cells were checked.</p>
-
     <p>The cells were  transferred into a 15 mL falcon and rinsed with 10 mL DMEM to collect all  remaining cells in the flask. The 15 mL falcon was filled with DMEM and the  culture spinned down at 1500 rpm  for 5  min at 4°C.</p>
+
     <p>The cells were  transferred into a 15 mL falcon and rinsed with 10 mL DMEM to collect all  remaining cells in the flask. The 15 mL falcon was filled with DMEM and the  culture spinned down at 1500 rpm  for 5  min at 4°C.</p>
-
     <p>The supernatant was  discarded and resuspended in 2 mL DMEM. The cells were splitted 1:3 which means  that 666 µL were taken from the suspension and transferred into a new culture  flask. After adding 20-25 mL DMEM the flask was incubated at 37°C and checked  on the 3rd / 4th day under the microscope.</p>
+
     <p>The supernatant was  discarded and resuspended in 2 mL DMEM. The cells were splitted 1:3 which means  that 666 Â&micro;L were taken from the suspension and transferred into a new culture  flask. After adding 20-25 mL DMEM the flask was incubated at 37°C and checked  on the 3rd / 4th day under the microscope.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 512: Line 512:
<li>place QIAquick spin column in 2ml collection tube</li>
<li>place QIAquick spin column in 2ml collection tube</li>
<li>centrifuge for 1min &rarr;discard flow-through</li>
<li>centrifuge for 1min &rarr;discard flow-through</li>
-
<li>add 750µl Buffer PE</li>
+
<li>add 750Â&micro;l Buffer PE</li>
<li>centrifuge for 1min  &rarr;discard flow-through</li>
<li>centrifuge for 1min  &rarr;discard flow-through</li>
<li>place column in 1,5 ml tube</li>
<li>place column in 1,5 ml tube</li>
-
<li>eluate with 50µl milipore water</li>
+
<li>eluate with 50Â&micro;l milipore water</li>
-
<li>incubate for 5min at 37°C, centrifuge for 1min</li>
+
<li>incubate for 5min at 37°C, centrifuge for 1min</li>
</ul>
</ul>
     </div>
     </div>
Line 578: Line 578:
   <tr>
   <tr>
     <th scope="row">Ampicilin</th>
     <th scope="row">Ampicilin</th>
-
     <td>Ampicilin (1000x) 100µl:1000ml <br /></td>
+
     <td>Ampicilin (1000x) 100Â&micro;l:1000ml <br /></td>
     <td>Final: 50&micro;g/ml</td>
     <td>Final: 50&micro;g/ml</td>
   </tr>
   </tr>
   <tr>
   <tr>
     <th scope="row">Canamycin</th>
     <th scope="row">Canamycin</th>
-
     <td>Canamycin (1000x) 100µl:100ml</td>
+
     <td>Canamycin (1000x) 100Â&micro;l:100ml</td>
     <td>Final: 50&micro;g/ml</td>
     <td>Final: 50&micro;g/ml</td>
   </tr>
   </tr>
     <tr>
     <tr>
     <th scope="row">Chloramphenicol</th>
     <th scope="row">Chloramphenicol</th>
-
     <td>Chloramphenicol (1000x) 100µl:100ml</td>
+
     <td>Chloramphenicol (1000x) 100Â&micro;l:100ml</td>
     <td></td>
     <td></td>
   </tr>
   </tr>
Line 615: Line 615:
     <td><table width="100%" border="1">
     <td><table width="100%" border="1">
       <tr>
       <tr>
-
         <td>10mM Hepes                     </td>
+
         <td>10mM Hepes                    </td>
         <td>1,19g</td>
         <td>1,19g</td>
       </tr>
       </tr>
Line 674: Line 674:
<div class="method">
<div class="method">
<fieldset class="microtiter">
<fieldset class="microtiter">
-
     <legend><a name="microtiter">Microtiter</a></legend>
+
     <legend><a name="microtiter">Microtiter-Assay</a></legend>
<div class="exp-content">
<div class="exp-content">
-
<p></p>
+
<ul class="comp">
-
    </div>
+
<li>1. Grow overnight cultures of <i>B. subtilis</i> in LB (+ antibiotic) or minimal medium S750</li>
 +
<li>2. Prepare medium, cells and Ag+ solutions depending on the assay
 +
<ul class="comp">
 +
<li>Cells: dilutions between 1:100 and 1:1000</li>
 +
<li>Ag+ dilutions: 10 mM to 0.1 nM</li>
 +
</ul>
 +
<li>3. Add 135 &micro;l Medium in every well of a 96 well plate (Premix of minimal medium and Ag+ dilution)</li>
 +
<li>4. Add 15 &micro;l cells to the medium</i>
 +
<li>5. Add 70 &micro;l mineral oil on top</li>
 +
<li>6.  Strains: As fluorescence is measured always include a positive and negative control</i>
 +
</ul>
 +
<br />
 +
<img src="https://static.igem.org/mediawiki/2014/0/05/MR_scheme_MTA.png" width="50%" />
 +
<br />
 +
<ul class="comp">
 +
<li>X = Medium (Control without cells)</li>
 +
<li>A-H = Different media</li>
 +
<li>1-9 = Strains</li>
 +
<li>10 = Wildtype (negative control)</li>
 +
<li>11 = GFP-Control</li>
 +
</ul>
 +
<br />
 +
<ul class="comp">
 +
<li>7. After the addition of media, cells and mineral oil it is important to check all wells for bubbles that have tob e removed with pipette tips. </li>
 +
<li>8. Start Plate reader program [120 Cycles]</li>
 +
<ul class="comp">
 +
<li>Shake 4 min</li>
 +
<li>Measurement OD<sub>450</sub></li>
 +
<li>Shake 1 min</li>
 +
<li>Measurement GFP</li>
 +
</ul>
 +
<li>9. Save data</li>
 +
<li>10. Evaluation: First step sort with R</li>
 +
<li>11. Evaluation: Second step excel</li>
 +
</ul>
 +
</div>
</fieldset>
</fieldset>
</div>
</div>

Latest revision as of 01:50, 18 October 2014

Notebook: Methods

Competent Cells Escherichia coli

1. Peparation for preculture

  • 50 µL Aliquod in 2x5ml LB each
  • incubation Overnight at 37°C

2. Preparation for main culture

  • 5ml of preculture in 250ml LB
  • incubation at 37°C until 0,5 OD (2h) →10min on ice

3. Making main culture competent

  • Pellets: 4x50ml in 4 Falcons
  • centrifugation at °C, 3500rpm, 15min
  • resuspending pellets in 4ml HTP* buffer for each Falcon (16ml total)
  • transfer new suspensions in 1 falcon → 10min on ice
  • centrifugation at 4°C, 3500rpm, 15min
  • resuspend pellet in 3ml HTP* buffer
  • add 225 µL DMSO
  • 50 µ aliquods → freeze in N2 (l)
  • store at -80°C
Transformation and Cultivation

1. Transformation

  • transfer 1 µL plasmid into aliquots for 10min on ice
  • 45sec. Heat shock at 42°C
  • 10 min on ice

2. Cultivation of transformed cells

  • add 700 µL LB to transformed cells
  • incubation for 1,5h at 37°C
  • centrifugation for 3min at 13000rpm
  • get off 600 µL per 1,5ml tube
  • resuspend pellet in left 100µL LB
  • plating on agar plate
  • incubation overnight
Expression Test: Induction + SDS-Gel

1. Picking colonies

  • inoculate 20 ml LB medium (sterile) containing 20µL antibiotics** and incubate until an OD of 0,6
  • 45sec. Heat shock at 42°C
  • 10 min on ice

2. Induction with IPTG/ Lactose

  • before induction: 0,7/ OD = sample volume
  • pellet + 80 µL water were resuspended in 20µL loading buffer (preinduction sample)
  • induction with 100 µL IPTG/ lactose at an OD of 0,6
  • incubation for 1,5h - 4 h max
  • after induction: 200 µL sample
  • pellet +80 µL water + 20 µL loading buffer resuspended (induction sample)
  • big volume gap because of decreased growth of bacteria
    after induction

3.Expression test with induced culture

  • transfer in 2x50ml falcons
  • centrifugation at 4°C, 3500rpm, 15min
  • washing pellets in 10ml buffer A
  • cracking cells with micro fluidizer
  • thermo centrifugation for 20 min – 4°C
  • 200 µL Ni-NTA beats + supernatant
  • 5min 4000 rpm → pellet
  • resuspended pellet in 500 µL Buffer A (low imidazole lv)
  • centrifugation 1 min – 4000rpm
  • pellet resuspended in buffer A
  • centrifugation 1 min – 4000rpm
  • pellet resuspended in 200 µL Buffer B (elution)
  • centrifugation 1min – 13000
  • 80µL supernatant (incl. protein)+ 20µL loading buffer

4. Expression test with induced culture → SDS-Gel

  • ladder – 5 µL
  • PI– preinduction sample - 10µL
  • I - induction sample -10 µL
  • E -elution sample -10 µL
Making Competent Bacillus subtilis

B. subtilis (PY79) is naturally competent (Albano et al., 1987). In order to increase the transformation rate, Bacillus can be grown in special media to enhance the competence. To achieve this, 20ml SPC-medium was inoculated with half a well grown LB-plate and incubated at 37°C until the cells reached the stationary phase. (Optical density OD550 nm does not change within a a timespan of 30 min) The cells were transfered into 100ml SPII-medium and incubated for 90min at 37°C. Afterwards, the cells were centrifuged (500 rpm, 15min, Rotor: BS4402/A Heraeus). The pellet was resuspended in 1ml glycerine (50% (v/v)). Samples were aliquoted and stored at -80°C.

For the transformation with plasmid-DNA 100µl cells and 5-7µl DNA (chromosomal DNA: 0,1-1µl DNA) were mixed andd incubated at 37°C for 30min. Afterwards, LB-plates (with antibiotics for selection) were inoculated with the culture and incubated at 30°C until colonies formed.

SPC-Medium:

T-Base (10x) 2 ml
D-Glucose Monohydrate 0,5% (w/v)
MgSO4 0,018% (w/v)
Caseinhydrolysate 0,025% (w/v)
Yeast-extract 0,2% (w/v)
ddH2O ad 20 ml

SPII-Medium

T-Base (10x)   10 ml
D-Glucose Monohydrat 50% (w/v)* 1 ml
MgSO4 1,2% (w/v) 7 ml
Caseinhydrolysate 1% (w/v) 1 ml
Yeast-extract 10% (w/v) 1 ml
CaCl2 0,1 M 500 μl
ddH2O ad   100 ml

10x T-Base:

(NH4)2SO4 150 mM
K2HPO4*3 H2O 1 M
KH2PO4 440 mM
Tri- Natriumcitrat di- Hydrat 39 mM

ddH2O ad 1000 ml

*Glucose was substituted with D-fructose in case one of the bacteria strains has a gene inserted under the xylose-promoter, since glucose does inhibit the uptake of xylose

Miniprep (Plasmid DNA Miniprep Kit II, Omega)
  • centrifuge at 10.000g for 1min
  • add 250µl Resuspension Buffer P1 per tube (incl. RNase), vortex
  • transfer suspension into new 1,5 ml tube
  • add 250µl Lysis Buffer P2 per tube, invert until lysate is clear (2-3min)
  • add 350µl Neutralisation Buffer N3 per tube
  • invert immediately (precipitate forms)
  • centrifuge 10min at 13.000 rpm
  • transfer supernatant onto column
  • centrifuge for 1min
  • add 500µl Washing Buffer PE (HBC Buffer) per tube
  • centrifugation for 1min throw filrate away
  • add 700µl DNA Wash Buffer per tube
  • centrifugation for 1min throw filrate away
  • add 30µl milipore per tube – let column sit for 2min
  • centrifuge for 2min
Gel Extraction (QIAquick Gel Extraction Kit)
  • band is cut out of the gel under UV light and transferred into a 1,5ml tube
  • weigh the gel with the tube
  • add QG (100mg gel → 100µl)
  • incubate for approx. 10min at 50-60°C, vortex to dissolve the gel
  • put spin colum into 2mlcollection tube
  • add dissolved gel onto column → centrifuge for 1min
  • discard flow-through
  • wash with 750µl Buffer PE → centrifuge for 1min
  • discard flow-through
  • place column in 1,5ml tube
  • elute with 50µl milipore water: incubate 5min at 37°C → centrifuge for 1min
pMAD-Transformation for Bacillus

1. Preculture for competent Bacillus subtilis

Aim:Preparation for mainculture the next day

5 mL of LB were inoculated with Bacillus WT3610 from an LB plate and incubated at 37°C overnight.

2. Transformation of competent Bacillus subtilis

Aim: transformation of plasmid into Bacillus subtilis WT3610

100 µL of overnight culture were added to 10 mL of MNGE-Medium and incubated till an OD of 1,1-1,3 at 37°C which could take 4-5h.4

After reaching OD of 1,1-1,3 400 µL of the culture were transformed with 1,5 µg plasmid. After 1h incubation at 37°C 100 µL Expression mix were added and incubated for 1h as well.

In the end the 500 µL attempt was plated out on MLS-X-Gal plates and incubated at 30 °C overnight until colonies could be seen.

3. Overnight culture of blue clones

Aim: transformation of plasmid into Bacillus subtilis WT3610

Colonies were grown on the plates with transformed plasmid. The blue/ white screening showed positive transformed blue clones. 3 clones of different morphology per plate were picked and used for inoculation of LB-MLS (4 mL LB, 4 µL Lincomycin, 4 µL Erythromycin). Incubation was carried out overnight at 30°C with the cultures.

4. First temperature shift

Aim: integration of pMAD-Insert into Bacillus chromosome via flanks

The overnight cultures were used to inoculate 10 mL LB MLS until the culture obtained an OD of 0,1. The cultures were incubated at 30°C for 2h.

Then the temperature was shifted to 42°C for 6h.

After the heat shock dilutions from 10-4 to 10-6 of each culture were plated out on MLS-X-Gal so that plates could be incubated overnight at 42°C.

5. Second temperature shift

Aim: flip out of the pMAD backbone

One blue colony per diluted clone was used to inoculate 4 mL LB. The cultures were incubated at 30°C for 6h and afterwards for 3h at 42°C.

Dilutions from 10-4­ to 10-6 were plated out on X-Gal plates WITHOUT MLS selection. The positive clones should not contain the resistance inside the backbone as well as the galactosidase. The plates were incubated at 42°C overnight.

6.1 selection of positive clones

Aim: checking the correct flip out of the pMAD backbone

From the dilution plates was one WHITE clone picked and transferred on a Master X-Gal Plate as well as on a MLS plate so that clones were proven for the right integration of the insert although flipping out the pMAD backbone.

The plates were incubated at 42°C overnight.

6.2 selection of positive clones

Aim: checking the correct flip out of the pMAD backbone

The white colonies grew on the X-Gal Master plates but not on MLS plates so the transformation seemed to be successful. The Backbone with the MLS resistance flipped out of the genome.

7. cPCR with checked clones

Aim: checking integration of constructs Hag-Spe and Hag-Spe-DARPin into B. s. genome

No clone was growing on MLS plates so the picked clones seemed to be positive. In order to check the correct integration of the domain constructs a cPCR with the picked clones on the X-Gal plates was done. The picked B.s. clones were cooked in 10 µL PBS for 5 min at 95°C.

Making Competent Bacillus with the High Salt/Low Salt Methode

modified after Harwood and Cutting 1990

LB-plates were inoculated with Bacillus subtilies and incubated at 37°C overnight.

3ml HS- (high-salt-) medium were inoculated with a single colony and incubated at 37°C overnight (rolling).

20ml preheated LS- (low salt-) medium was inoculated with 1ml HS-culture and incubated at 30°C for 3h at 100rpm in a water bath.

10 x S-Base

(NH4)2SO4 2g
K2HPO4 14g
KH2PO4 6g
Natriumcitrat* 2 H2O 1g
aqua dest. ad 100ml
After being autoclaved 0,1ml 1M MgSO4 (sterile filtered) were added  

HS-Medium

aqua dest. 74,5 ml
10 x S-Base 10 ml
50% Glucose 1 ml
Tryptophan 5 mg/ml 1 ml
Phenylalanin 3 mg/ml 1,5 ml
Casein-Hydrolysat 2 % 1 ml
Yeast-extract 10 % 1 ml
Arginin 8 % + Histidin 0,4 % 10 ml
Solutions were sterile filtered and mixed under sterile conditions  

LS-Medium

aqua dest. 17,2 ml
10 x S-Base 2 ml
50% Glucose 0,2 ml
Tryptophane 5 mg/ml 20 µl
Phenylalanine 3 mg/ml 30 µl
Casein-Hydrolysat 2 % 0,1 ml
Yeast-extract 10 % 0,2 ml
Spermin 50 mM 0,2 ml
MgCl2 1M 50 µl
Solutions were prepared directly before use and mixed under sterile conditions  

Transformation

Prepare DNA-Solution (1-5µg plasmid DNA or 10-20µg chromosomal DNA) in eppendorf cup.
Add 1ml LS-culture, incubate the transformation samples at 37°C for 2h an a thermomixer/roller.
Centrifuge the transformation samples fpr 20sec, discard some of the supernatant and resuspend the cells.
Incubate selective plates.

(Harwood, C.R., and Cutting, S.M. (1990) Molecular biological methods for Bacillus. John Wiley and Sons Ltd., Chichester, England.)

Transformation of Bacillus subtilis with Spizizens minimal medium
  • 10 ml LB inoculated with single colony as preculture over night at 37°C
  • Inoculate 10 ml Spizizens minimal medium with the preculture to an OD600 of 0.1
  • Incubation at 37°C until OD600 1 more or less
  • Addition of 5 µg plasmid DNA/5-10 µg of chromosomal DNA to 1 ml of culture (in a test tube)
  • Incubation at 37°C for 2 h
  • Plate out 200 µl and 500 µl on different plates containing selecting antibiotics and incubate the plates at 37°C overnight
Purification of Ni-NTA column

The following steps were performed for the Ni-NTA Column:

  • equilibration with Buffer A 10 min
  • taking 40 µL supernatant (load)+ 10 µL SDS-Buffer - L-sample
  • 50 mL load on column
  • taking 40 µL of flow through + 10 µL SDS-buffer - FT-sample
  • first washing with 25ml Buffer A (half the load)
  • taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-sample
  • equilibrate with Buffer B (output pipe off the column into glas, input into B)
  • hanging pipe on column, 20 ml Elution - E
  • taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E- sample
  • SDS-PAGE analysis

Regeneration of column

  • 10min water
  • 10 min EDTA
  • 10min water
  • 10min NiSO4
  • 10min water
Splitting of Caco-2-cells

Caco-2-cells require DMEM (Dulbecco’s Modified Eagle Medium) with 20% FCS and L-Glutamine.

A centrifuge was set on 4°C.

The medium of the culture flask was discarded from the side opposite to the adherent cells and washed with 1X PBS from the opposite site as well.

The PBS was aspirated with a glass pipette until the supernatant was clear. After adding 1-2 mL 1x trypsin from the opposite of the adherent cells the flask was incubated for 5-10 min at 37°C depending on how fast the cells come of from the ground. Under the microscope the free floating cells were checked.

The cells were transferred into a 15 mL falcon and rinsed with 10 mL DMEM to collect all remaining cells in the flask. The 15 mL falcon was filled with DMEM and the culture spinned down at 1500 rpm for 5 min at 4°C.

The supernatant was discarded and resuspended in 2 mL DMEM. The cells were splitted 1:3 which means that 666 µL were taken from the suspension and transferred into a new culture flask. After adding 20-25 mL DMEM the flask was incubated at 37°C and checked on the 3rd / 4th day under the microscope.

Purification of PCR Products (QIAquick Gel Extraction Kit) short protocol

short protocol without gel extraction

  • add 3 volumes or QG Buffer to tubes and 1 volume isopropanol
  • place QIAquick spin column in 2ml collection tube
  • centrifuge for 1min →discard flow-through
  • add 750µl Buffer PE
  • centrifuge for 1min →discard flow-through
  • place column in 1,5 ml tube
  • eluate with 50µl milipore water
  • incubate for 5min at 37°C, centrifuge for 1min
Annealing with PNK Inactivation
Step Temperature °C Time
1 37 1h
2 65 20 min
3 95 5 min
4 95-4 1 min/1°C
5 4 Infinite
Medium
LB-medium Concentration Final
Ampicilin Ampicilin (1000x) 100µl:1000ml
Final: 50µg/ml
Canamycin Canamycin (1000x) 100µl:100ml Final: 50µg/ml
Chloramphenicol Chloramphenicol (1000x) 100µl:100ml
Buffer
Buffer Ingredients
-2.0 Buffer 2.0 (10x) + BSA (10mg/ml -10) → BSA end concentration 100µg/ml (0,1µg/µl)
HTP (for 500ml)
10mM Hepes 1,19g
15mM CaCl2 x2H20 1,1g
250mM KCl 9,32g
55mM MnCl2 x 4 H2O 5,44g
Buffer A 20mM HEPES
250mM NaCl
20mM KCl
20 mM MgCl
40 Imidazole
pH 8,0
Buffer B 20mM HEPES
250mM NaCl
20mM KCl
20 mM MgCl
500 mM Imidazole
pH 8,0
GeFi Buffer 2mM HEPES
20mM NaCl
2mM KCl
2 mM MgCl
pH 7,5
Inclusion Body Wash Buffer 100 mM NaCl
50 mM Tris
pH 8.0
0.5% Triton X-100
Microtiter-Assay
  • 1. Grow overnight cultures of B. subtilis in LB (+ antibiotic) or minimal medium S750
  • 2. Prepare medium, cells and Ag+ solutions depending on the assay
    • Cells: dilutions between 1:100 and 1:1000
    • Ag+ dilutions: 10 mM to 0.1 nM
  • 3. Add 135 µl Medium in every well of a 96 well plate (Premix of minimal medium and Ag+ dilution)
  • 4. Add 15 µl cells to the medium
  • 5. Add 70 µl mineral oil on top
  • 6. Strains: As fluorescence is measured always include a positive and negative control


  • X = Medium (Control without cells)
  • A-H = Different media
  • 1-9 = Strains
  • 10 = Wildtype (negative control)
  • 11 = GFP-Control

  • 7. After the addition of media, cells and mineral oil it is important to check all wells for bubbles that have tob e removed with pipette tips.
  • 8. Start Plate reader program [120 Cycles]
    • Shake 4 min
    • Measurement OD450
    • Shake 1 min
    • Measurement GFP
  • 9. Save data
  • 10. Evaluation: First step sort with R
  • 11. Evaluation: Second step excel

Retrieved from "http://2014.igem.org/Team:Marburg:Project:Notebook:Methods"