NEED PROTOCOL: PCRing genes from E.Coli

Rogier rogierNOroSPAM at
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


get money for reviews on kits, enzymes, and other tools for
molecular biology:

(if you sign up thru this link, biowire gives you a laserpointer
and I get 5 bucks from them)

any ads below this line are inserted by I'm innocent!


Got questions?  Get answers over the phone at
Up to 100 minutes free!

More information about the Microbio mailing list