Team:EPF-Lausanne/Protocol/Transformation

(Difference between revisions)

Revision as of 16:45, 10 July 2012

Thaw the competent E.coli (DH5alpha) cells on ice (not in hands!)
As soon as it is thawed, add 50µl of the cells to the DNA (~50-100ng of pure plasmid, or some 2 µl usually)
Let it rest on ice for 20-30 min. Meanwhile, put agar plate (with correct antibiotic) at 37°C for prewarming.
Put the tube with DNA+E.coli at 42°C for 45 sec-1 min (heat shock)
Add 400ul of LB broth and place at 37°C for 20-30min (shaking)
Spread the cells on the prewarmed plate (and let it dry)
Incubate the plate upside-down at 37°C for ~14-15h (leaving it more than 16h decreases the plasmid quality)

@@ Line 9: / Line 9: @@
 # Thaw the competent E.coli (DH5alpha) cells on ice (not in hands!)
-# As soon as it is thawed, add 50ul of the cells to the DNA (~50-100ng of pure plasmid)
+# As soon as it is thawed, add 50µl of the cells to the DNA (~50-100ng of pure plasmid, or some 2 µl usually)
 # Let it rest on ice for 20-30 min. Meanwhile, put agar plate (with correct antibiotic) at 37°C for prewarming.
 # Put the tube with DNA+E.coli at 42°C for 45 sec-1 min (heat shock)