Team:Paris Bettencourt/Notebook/Interlab Study

From 2014.igem.org

(Difference between revisions)
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<h4>July </h4>
<h4>July </h4>
<h5 id="date">July 4th</h5>
<h5 id="date">July 4th</h5>
-
<p id="text"><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;"><div><div style="font-size: 15px;"><u>We will participate in the iGEM interlab study: https://2014.igem.org/Tracks/Measurement/Interlab_study</u></div><div><br clear="none"/></div><div>The three specific devices teams are required to measure fluorescence data for are given below.</div><div><br clear="none"/></div><div>Existing device: <strong>BBa_I20260 (J23101-B0032-E0040-B0015)</strong> in the <strong>pSB3K3</strong> vector.</div><ol><li>Kit location
+
<p id="text">We will participate in the<a href=”https://2014.igem.org/Tracks/Measurement/Interlab_study” >  iGEM interlab study</a></p>
-
<ol><li>Plate 4, Well 18A</li></ol></li></ol><div>E.Coli  <strong>NEBTurbo</strong> competent cells</div><p>To use the DNA in the Distribution Kit you may follow these instructions:</p><ol><li>With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want.</li><li>Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.</li><li><a href="http://parts.igem.org/Help:Transformation_Protocol" shape="rect" target="_blank" title="Help:Transformation Protocol"><span style="color:rgb(0,102,204);"><span>Transform</span></span></a> 1 or 2uL of the resuspended DNA into your desired competent cells,</li></ol><div><u style="font-size: 15px;">Heat Shock Transformation of <em>E. coli</em></u></div><p>This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.</p><h5>Note: Never vortex competent cells. Mix cells by gentle shaking.</h5><ol><li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.</li><li>Place 20 ul of cells in a pre-chilled Eppendorf tube.
+
-
<ul><li><span style="text-decoration: underline;">For an Intact Vector:</span> Add 0.5 ul or less to the chilled cells</li></ul></li><li>Mix gently by flicking the tube.</li><li>Chill on ice for 10 minutes. <em>This step is optional, but can improve yields when transforming a ligation product.</em></li><li>Heat shock at 42 °C for 30 seconds.</li><li>Return to ice for 2 minutes.</li><li>Add 200 ul LB medium and recover the cells by shaking at 37 °C.Kanamycin: 120 minutes</li><li>Plate out the cells on selective LB.Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.</li><li>Incubate at 37 °C. Transformants should appear within 12 hrs.</li></ol><div><span style="background-color:rgb(255, 250, 165);-evernote-highlight:true;"><u>Result:</u></span> Bacteria grow and made big ans small colonies</div><div><br clear="none"/></div></div><div>     4.Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.</p>
+
<p><b><u>Device 1</b></u></p>
 +
 
 +
<p>Existing device: </b>BBa_I20260 (J23101-B0032-E0040-B0015)</b> in the pSB3K3 vector.</p>
 +
 
 +
<ul> Kit location
 +
 
 +
<li>Spring 2014 Plate 4, Well 18A</li>
 +
 
 +
</ul>
 +
<p>We use <b>NEB Turbo</b> cells to transform with the Biobrick</p>
 +
<p><u>Biobrick extraction (iGEM protocol)</u></p>
 +
<p id="kit">To use the DNA in the Distribution Kit you may follow these instructions:</p>
 +
<ol>
 +
<li> With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want.</li>
 +
<li> Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.</li>
 +
 
 +
</ol></p>
 +
<p>Then we followed our protocol for <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1">Heat Shock transformation of <i>E.coli</i></a></p>
 +
 
<h5 id="date">July 5th</h5>
<h5 id="date">July 5th</h5>
-
<p id="text">Bacterias plated yesterday growed and made big and small colonies</font></h2><div>I picked one small and one big colony fom one plate in order to meake the Glycerol stock</p>
+
<p id="text">Bacterias plated yesterday growed and made big and small colonies.I picked one small and one big colony from one plate in order to make liquid culture:
-
<p id="text"><u>Glycerol Stocks</u></br>
+
<ol>
-
<ol><li>Pick Single colonies from agar plates</li><li>Innoculate 5ml LB broth overnight.</li></ol></p>
+
<li>5mL of LB</li>
-
<p id="text">Text</p>
+
<li>5um Chlorophenicol</li>
 +
<li>Inoculate a single colony</li>
 +
</ol>
 +
</p>
 +
 
 +
<h5 id="date">July 6th</h5>
 +
<p id="text">I made a glycerol stock from the liquid culture following the standard protocol of the <a href=https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">Glycerol stock </a> and labelled it with a number G.22</p>
 +
</br>
 +
</br>
 +
<h5 id="date">July 7th</h5>
 +
<p id="text"><u><b>Devices 2 and 3 </u></b></p>
 +
<p> New device to be built by the iGEM team: BBa_J23101 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone
 +
</br>
 +
<ol>Kit locations (Spring 2014)
 +
</br>
 +
<li> BBa_J23101 (called BBa_K823005 when in pSB1C3): Plate 1, Well 20K </li>
 +
<li>BBa_E0240 (in pSB1C3): Plate 2, Well 24B</li>
 +
</ol>
 +
</p>
 +
 
 +
 
 +
New device to be built by the iGEM team: BBa_J23115 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone
 +
</br>
 +
<ol>Kit locations (Spring 2014)
 +
</br>
 +
<li> BBa_J23115 (called BBa_K823012 when in pSB1C3): Plate 1, Well 22I</li>
 +
<li>BBa_E0240 (in pSB1C3): Plate 2, Well 24B</li>
 +
</br>
 +
</ol>
 +
</p>
 +
 
 +
<p> I followed <a href=kit>iGEM Distribution Kit instructions </a> and <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1">Heat Shock transformation of <i>E.coli</i></a>
 +
</p>
 +
</br>
 +
 
 +
<h5 id="date">July 8th</h5>
 +
 +
<p id="text"><ul>Successful transformations:
 +
<li> BBa_E0240 </li>
 +
<li>BBa_K823012</li></ul></p>
 +
<p id="text"><ul>Unsuccessful transformation:
 +
<li> BBa_K823005</li>
 +
</ul></p>
 +
 +
<p> For the <u>successful transformations</u> I followed the <a href=https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">Glycerol stock </a> protocol </p>
 +
 +
<p>For the <u> unsuccessful transformation </u> I transformed NEB turbo again with the rest of Biobrick BBa_K823005 following again <a href=kit>iGEM Distribution Kit instructions </a> and <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1">Heat Shock transformation of <i>E.coli</i></a></p>
</br>
</br>
-
</div>
+
 
 +
<h5 id="date">July 9th</h5>
 +
 +
<p> I finished the <a href=https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">Glycerol stock </a> protocol and labelled G.23 for BBa_K823012 and G.24 for BBa_E0240</p>
 +
 +
<p><b>BBa_K823005 </b> successfully transformed! I followed the <a href=https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">Glycerol stock </a> protocol </p>
 +
 +
<p><u>Minipreps</u>
 +
</br>
 +
Following the standard  <a href=https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot4">protocol fot the miniprep  </a></br>
 +
I measured DNA content with the nanodrop. </br>
 +
<u>Digestion analysis</u>: (Clovis's recipe)</br>
 +
- 5 ug plasmid (given 211ng of BBa_E0240 - <b>15uL</b> // given 387ng of BBa_K823012  - <b>30uL</b>  </br>
 +
- 5 ul FD Buffer</br>
 +
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823012) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)</br>
 +
- complete with H2O</br>
 +
 
 +
(Final volume of 50 uL)</br>
 +
</p>
 +
<p><u>Result:</u>DNA wasn't cut. Possible causes: </br> - wrongly calculated volumes (BBa_K823012 shouldn't be 30uL) </br> - too short digestion time</p>
 +
 +
</br>
 +
 
 +
<h5 id="date">July 10th</h5>
 +
<p>Finished the <a href=https://2014.igem.org/Team:Paris_Betmisslabelledtencourt/Protocols#prot8">Glycerol stock </a> protocol and labelled it G.25</p>
 +
<p><u> Minipreps</u>
 +
Following the standard  <a href=https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot4">protocol fot the miniprep  </a></br>
 +
I measured DNA content with the nanodrop. </br>
 +
<u>Digestion analysis</u>: (Clovis's recipe)</br>
 +
- 5 ug plasmid </br>
 +
- 5 ul FD Buffer</br>
 +
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823012) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)</br>
 +
- complete with H2O</br>
 +
 
 +
(Final volume of 50 uL)</br>
 +
In the final 30uL of the miniprep of  BBa_K823005  I didn't have enough of DNA.
 +
</p>
 +
 +
</br>
 +
 
 +
<h5 id="date">July 11th</h5>
 +
<p><u> Minipreps</u> [yes, again!]
 +
Following the standard  <a href=https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot4">protocol fot the miniprep  </a></br>
 +
I measured DNA content with the nanodrop. </br>
 +
<u>Digestion analysis</u>: (Clovis's recipe)</br>
 +
- 5 ug plasmid </br>
 +
- 5 ul FD Buff(given 211ng of BBa_E0240 - <b>21uL</b> // given 144ng and 120ng of BBa_K823012 - <b>35uL</b> // given 273ng of BBa_K823005 - <b>20uL</b>  </br>
 +
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823012 and BBa_K823005) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)</br>
 +
- complete with H2O</br>
 +
 
 +
(Final volume of 50 uL)</br>
 +
 
 +
<u>Result:</u> The gel showed that I mislabelled the constructs. There were 3 promoters (big and very small fragments) </br> I will restart the experiment from the Biobricks
 +
 
 +
</p>
 +
 
 +
</p>
 +
 +
 
 +
 
 +
</br> </div>
<div id="August">
<div id="August">
<h4>August</h4>
<h4>August</h4>

Revision as of 09:28, 21 August 2014

Paris Bettencourt 2014



Notebook

Interlab Study

July

July 4th

We will participate in the iGEM interlab study

Device 1

Existing device: BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector.

    Kit location
  • Spring 2014 Plate 4, Well 18A

We use NEB Turbo cells to transform with the Biobrick

Biobrick extraction (iGEM protocol)

To use the DNA in the Distribution Kit you may follow these instructions:

  1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want.
  2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.

Then we followed our protocol for Heat Shock transformation of E.coli

July 5th

Bacterias plated yesterday growed and made big and small colonies.I picked one small and one big colony from one plate in order to make liquid culture:

  1. 5mL of LB
  2. 5um Chlorophenicol
  3. Inoculate a single colony

July 6th

I made a glycerol stock from the liquid culture following the standard protocol of the Glycerol stock and labelled it with a number G.22



July 7th

Devices 2 and 3

New device to be built by the iGEM team: BBa_J23101 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone

    Kit locations (Spring 2014)
  1. BBa_J23101 (called BBa_K823005 when in pSB1C3): Plate 1, Well 20K
  2. BBa_E0240 (in pSB1C3): Plate 2, Well 24B

New device to be built by the iGEM team: BBa_J23115 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone
    Kit locations (Spring 2014)
  1. BBa_J23115 (called BBa_K823012 when in pSB1C3): Plate 1, Well 22I
  2. BBa_E0240 (in pSB1C3): Plate 2, Well 24B

I followed iGEM Distribution Kit instructions and Heat Shock transformation of E.coli


July 8th

    Successful transformations:
  • BBa_E0240
  • BBa_K823012

    Unsuccessful transformation:
  • BBa_K823005

For the successful transformations I followed the Glycerol stock protocol

For the unsuccessful transformation I transformed NEB turbo again with the rest of Biobrick BBa_K823005 following again iGEM Distribution Kit instructions and Heat Shock transformation of E.coli


July 9th

I finished the Glycerol stock protocol and labelled G.23 for BBa_K823012 and G.24 for BBa_E0240

BBa_K823005 successfully transformed! I followed the Glycerol stock protocol

Minipreps
Following the standard protocol fot the miniprep
I measured DNA content with the nanodrop.
Digestion analysis: (Clovis's recipe)
- 5 ug plasmid (given 211ng of BBa_E0240 - 15uL // given 387ng of BBa_K823012 - 30uL
- 5 ul FD Buffer
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823012) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
- complete with H2O
(Final volume of 50 uL)

Result:DNA wasn't cut. Possible causes:
- wrongly calculated volumes (BBa_K823012 shouldn't be 30uL)
- too short digestion time


July 10th

Finished the Glycerol stock protocol and labelled it G.25

Minipreps Following the standard protocol fot the miniprep
I measured DNA content with the nanodrop.
Digestion analysis: (Clovis's recipe)
- 5 ug plasmid
- 5 ul FD Buffer
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823012) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
- complete with H2O
(Final volume of 50 uL)
In the final 30uL of the miniprep of BBa_K823005 I didn't have enough of DNA.


July 11th

Minipreps [yes, again!] Following the standard protocol fot the miniprep
I measured DNA content with the nanodrop.
Digestion analysis: (Clovis's recipe)
- 5 ug plasmid
- 5 ul FD Buff(given 211ng of BBa_E0240 - 21uL // given 144ng and 120ng of BBa_K823012 - 35uL // given 273ng of BBa_K823005 - 20uL
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823012 and BBa_K823005) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
- complete with H2O
(Final volume of 50 uL)
Result: The gel showed that I mislabelled the constructs. There were 3 promoters (big and very small fragments)
I will restart the experiment from the Biobricks


August

Date 1

Text

Text

Date 2

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Text


September

Date 1

Text

Text

Date 2

Text

Text


October

Date 1

Text

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Date 2

Text

Text


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