NEED PROTOCOL: PCRing genes from E.Coli

Rogier rogierNOroSPAM at bio.vu.nl.invalid
Tue Jul 4 13:12:34 EST 2000


Quick'n'dirty PCRs of small multicopy plasmids in bugs usually
don't work for big single/double copy genomes.
E. coli's single circular chromosome is about a 1000 times bigger
than the average cloning vector, and it's copy number hundreds of
times lower.

This is what worked in my hands (both for yeast and E. coli):

put a colony in 5x PCR buffer
boil for 5 min. (do not microwave)
dilute 1:5 into a standard PCR reaction.

Next trick: which enzyme to use?
Taq is sloppy, HiFi polymerases are less efficient. Taq/HiFi
mixes (sold by many companies) are sloppy and inefficient.
Solution: start with Taq polymerase for 5 cycles, then dilute the
Taq reaction 1:10 into a HiFi-polymerase reaction.
The error rate of Taq is not a problem after 5 cycles, but this
pre-PCR gives you a clean-and-easy template for the picky
Pfu/Vent/DeepVent/etc. polymerases.

Rogier Stuger
MicFizz, Free U, Amsterdam
rogier AT bio.vu.nl

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