Team:EPF-Lausanne/Protocol/Transformation

(Difference between revisions)

Revision as of 12:49, 30 June 2012

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)
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 10: / Line 10: @@
 # 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)
-# Let it rest on ice for 20-30 min. Meanwhile, put agar plate (with correct antibiotic)
+# Let it rest on ice for 20-30 min. Meanwhile, put agar plate (with correct antibiotic) at 37°C for prewarming.
-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
+# Incubate the plate upside-down at 37°C for ~14-15h (leaving it more than 16h decreases the plasmid quality)
-the plasmid quality)
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