Team:Groningen/protocols/Transformation EC

From 2013.igem.org

(Difference between revisions)
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<h3>Steps:</h3>
<h3>Steps:</h3>
<ol>
<ol>
-
<li>Prepare plates with the correct selection marker.</li>
+
<li>Prepare plates with the correct antibiotic selection marker.</li>
-
<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>
+
<li>Prechill the eppendorf tubes on ice.</li>
-
<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>Add 10μl ligation reaction to a sterile tube.</li>
 +
<li>Remove a tube of frozen competent cells from the storage (-80°C freezer) and thaw it on ice (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>Add 10μl ligation reaction to the competent cells.</li>
-
<li>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).</li>
+
<li>Gently flick the tubes to mix and place them on ice for 30 minutes.</li>
 +
<li>Heat-shock the cells for 60 seconds in a water bath at 42°C (do not shake and make sure the content of the tube is all at the bottom).</li>
<li>Immediately return the tubes to ice for 2 minutes.</li>
<li>Immediately return the tubes to ice for 2 minutes.</li>
-
<li>Add 900μl room-temperature LB broth to the tubes containing cells.</li>
+
<li>Add 1ml LB broth (RT) to the reaction mixture.</li>
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<li>Incubate for 1.5 hours at 37°C with shaking (~150rpm).</li>
+
<li>Incubate for at 37°C 1 hour with shaking (~150rpm).</li>
-
<li>Plate 200μl of each transformation culture on LB plates.</li>
+
<li>Centrifuge the tubes at 14000 rpm for 1min.</li>
 +
<li>Empty the tube until about 200μl supernatant is left.</li>
 +
<li>Resuspend the pellet in the supernatant.</li>
 +
<li>Plate 200μl of each transformation culture on LB agar plates with an ampicillin resistant marker.</li>
<li>Incubate the plates overnight (16–24 hours) at 37°C.</li>
<li>Incubate the plates overnight (16–24 hours) at 37°C.</li>
-
<li>Store of plates at 4°C (after 37°C overnight incubation).</li>
+
<li>Afterwards plates can be stored 4°C.</li>
</ol>
</ol>

Revision as of 17:56, 27 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 antibiotic selection marker.
  2. Prechill the eppendorf tubes on ice.
  3. Add 10μl ligation reaction to a sterile tube.
  4. Remove a tube of frozen competent cells from the storage (-80°C freezer) and thaw it on ice (about 5 minutes). Mix the cells by gently flicking the tube. Avoid excessive pipetting, as the competent cells are extremely fragile.
  5. Carefully transfer 50μl of cells into each tube prepared in Step 2.
  6. Add 10μl ligation reaction to the competent cells.
  7. Gently flick the tubes to mix and place them on ice for 30 minutes.
  8. Heat-shock the cells for 60 seconds in a water bath at 42°C (do not shake and make sure the content of the tube is all at the bottom).
  9. Immediately return the tubes to ice for 2 minutes.
  10. Add 1ml LB broth (RT) to the reaction mixture.
  11. Incubate for at 37°C 1 hour with shaking (~150rpm).
  12. Centrifuge the tubes at 14000 rpm for 1min.
  13. Empty the tube until about 200μl supernatant is left.
  14. Resuspend the pellet in the supernatant.
  15. Plate 200μl of each transformation culture on LB agar plates with an ampicillin resistant marker.
  16. Incubate the plates overnight (16–24 hours) at 37°C.
  17. Afterwards plates can be stored 4°C.