PCR directly on whole E. coli

Benoit_Hebert at IAF.UQUEBEC.CA Benoit_Hebert at IAF.UQUEBEC.CA
Tue May 24 22:13:51 EST 1994


Charlie Hoffman (HOFFMACS at hermes.bc.edu) wrote:
> I'd like to know now
> whether or not you can do the same with E. coli to screen transformants
> for your plasmid of interest?  If so, do you have to be careful not to
> add to many cells?  How many?

all the replies that you received had similar methods as you can see. There
are a few things to remember:

1- media will cause problems
2- a colony in 50 microL of saline is OK, take 2 microL for the PCR
3- 1 microL of O/N culture is good too
4- keep low annealing T even if you get extra bands
5- using the universal (forward) sequencing primer and universal reverse 
primers (e.g. in pUC18,19,...), you can verify the size of the insert. If your 
insert is small and there is not much difference with the distance between 
your cloning sites, I have designed a primer (in the beta-gal region) which 
will increase the size and will make it easier to distinguish the positive 
clones. Sequence available on request (give me time to look it up!)
6- by using at least one internal primer, you verify the presence of the 
correct insert and its orientation in one shot

have fun!

Benoit Hebert

========================================
Benoit_Hebert at iaf.uquebec.ca

Armand-Frappier Institute
Virology Research Center
Laval, Quebec, Canada    H7N 4Z3
phone: (514) 687-5010, ext. 4636
fax: (514) 686-5626
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