Team:INSA Toulouse/contenu/lab practice/notebook/protocols/charac recomb

From 2013.igem.org

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   <h2 class="title2">Protocols</h2>
   <h2 class="title2">Protocols</h2>
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   <h3 class="title3">DNA Kit Plate Instructions (iGEM protocol)</h2>
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   <h3 class="title3">In vitro recombinase characterization protocol</h2>
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   <p class="texte">Before you use the DNA in the Distribution Kit Plates, be sure to test the efficiency of your competent cells with the Transformation Efficiency Kit.
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To use the DNA in the Distribution Kit, follow these instructions: <br><br>
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   <p class="texte"><b>Recombinase overexpression and extraction</b><br>
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Note: <i>There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/µl.</i></p>
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<div class="list">
<div class="list">
     <ol >
     <ol >
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       <li>With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.</li>
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       <li>The E.coli K12-DH5.1 containing recombinase and Cloramphenicol resistance genes were grown in variable amount of Chloramphenicol LB culture medium.</li>
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       <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. We recommend that you do not use TE to resuspend the dried DNA.</li>
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       <li>After 4h growth at 37°C, the culture was centrifuged for 3 min at 13000 rpm.</li>
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       <li>Transform 1ul of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.</li>
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       <li>The cell pellets were resuspended in 500µl <b>Rec buffer</b> containing 20 mM Tris, pH 7.5, 10mM EDT1, 25mM NaCl, 10mM Spermidine, 1mM DTT and 0.1g.mol-1 BSA.</li>
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       <li>Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.</li>
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       <li>The cells were lysed by sonication for three bursts of 30s.</li>
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       <li>Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.</li>
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       <li>After centrifugation for 15 min at 13000 rpm, 4°C, the supernatant containing proteins (recombinase) was collected and incubated on ice.</li>
     </ol>
     </ol>
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<br></div>
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  <p class="texte"><b>In vitro recombination test</b><br>
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<div class="list">
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    <ol >
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      <li>Recombination reactions were assembled on ice with recombinase protein extraction solution and a variable amount of solution containing <b>Rec buffer</b> and the gate plasmid.</li>
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      <li>Reactions were incubated at 37°C for up to 2 hours and heat inactivated at 75°C for 10min.</li>
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      <li>Reactions were transformed into E.coli K12-DH5.1.</li>
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      <li>Spread out the cells on Kanamycine LB agar plate.</li>
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      <li>The plate was incubated overnight at 37°C.</li>
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    </ol>
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<br>
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<br></div>
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  <p class="texte">Note: For Xor1 gate characterization, the in vitro recombination test was performed at different Bxb1 concentrations (100µl and 20µl of Bxb1 extraction solution) and different hours of reactions (2 hours and 5 hours).<br>
   <p class="texte"><i>* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.</i><br>
   <p class="texte"><i>* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.</i><br>

Revision as of 16:14, 20 September 2013

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Notebook

Protocols

In vitro recombinase characterization protocol

Recombinase overexpression and extraction

  1. The E.coli K12-DH5.1 containing recombinase and Cloramphenicol resistance genes were grown in variable amount of Chloramphenicol LB culture medium.
  2. After 4h growth at 37°C, the culture was centrifuged for 3 min at 13000 rpm.
  3. The cell pellets were resuspended in 500µl Rec buffer containing 20 mM Tris, pH 7.5, 10mM EDT1, 25mM NaCl, 10mM Spermidine, 1mM DTT and 0.1g.mol-1 BSA.
  4. The cells were lysed by sonication for three bursts of 30s.
  5. After centrifugation for 15 min at 13000 rpm, 4°C, the supernatant containing proteins (recombinase) was collected and incubated on ice.


In vitro recombination test

  1. Recombination reactions were assembled on ice with recombinase protein extraction solution and a variable amount of solution containing Rec buffer and the gate plasmid.
  2. Reactions were incubated at 37°C for up to 2 hours and heat inactivated at 75°C for 10min.
  3. Reactions were transformed into E.coli K12-DH5.1.
  4. Spread out the cells on Kanamycine LB agar plate.
  5. The plate was incubated overnight at 37°C.


Note: For Xor1 gate characterization, the in vitro recombination test was performed at different Bxb1 concentrations (100µl and 20µl of Bxb1 extraction solution) and different hours of reactions (2 hours and 5 hours).

* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.
Note: There is not enough DNA in each well to perform anything but transformations.

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