Team:Groningen/protocols/Transformation EC

From 2013.igem.org

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<h3>Steps:</h3>
<h3>Steps:</h3>
<ol>
<ol>
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<li>Prepare plates with the correct selection marker (which are usually kept in the fridge or in the freezer depending on the stability of the molecule) for each ligation reaction.</li>
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<li>Prepare plates with the correct selection marker.</li>
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<li>Centrifuge the tubes containing the ligation reactions to collect the contents at the bottom. Add the 5μl ligation reaction to a sterile (17 × 100mm) polypropylene tube or a 1.5ml microcentrifuge tube on ice (see Note 1).</li>
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<li>Centrifuge the tubes containing the DNA to be transformed to collect the content at the bottom. Add the 2μl ligation reaction to a sterile tube.</li>
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<li>Remove tube(s) of frozen Competent Cells from the storage (-80 °C freezer) and place in ice until just thawed (about 5 minutes). Mix the cells by gently flicking the tube. Avoid excessive pipetting, as the competent cells are extremely fragile.</li>
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<li>Remove tube of frozen Competent Cells from the storage (-80 °C freezer) and place in ice until just thawed (about 5 minutes). Mix the cells by gently flicking the tube. Avoid excessive pipetting, as the competent cells are extremely fragile.</li>
<li>Carefully transfer 50μl of cells into each tube prepared in Step 2.</li>
<li>Carefully transfer 50μl of cells into each tube prepared in Step 2.</li>
<li>Gently flick the tubes to mix and place them on ice for 20 minutes.</li>
<li>Gently flick the tubes to mix and place them on ice for 20 minutes.</li>

Revision as of 13:38, 26 July 2013

E. Coli transformation protocol

Materials:

  • LB plates with selection markers (antibiotics, inducers,…)
  • LB broth
  • Eppendorf tubes (preferably 2 ml)
  • Spreaders
  • DNA to be transformed

Steps:

  1. Prepare plates with the correct selection marker.
  2. Centrifuge the tubes containing the DNA to be transformed to collect the content at the bottom. Add the 2μl ligation reaction to a sterile tube.
  3. Remove tube of frozen Competent Cells from the storage (-80 °C freezer) and place in ice until just thawed (about 5 minutes). Mix the cells by gently flicking the tube. Avoid excessive pipetting, as the competent cells are extremely fragile.
  4. Carefully transfer 50μl of cells into each tube prepared in Step 2.
  5. Gently flick the tubes to mix and place them on ice for 20 minutes.
  6. Heat-shock the cells for 80 seconds in a water bath at exactly 37°C (do not shake and make sure the content of the tube is all at the bottom).
  7. Immediately return the tubes to ice for 2 minutes.
  8. Add 900μl room-temperature LB broth to the tubes containing cells.
  9. Incubate for 1.5 hours at 37°C with shaking (~150rpm).
  10. Plate 200μl of each transformation culture on LB plates.
  11. Incubate the plates overnight (16–24 hours) at 37°C.
  12. Store of plates at 4°C (after 37°C overnight incubation).