NEED PROTOCOL: PCRing genes from E.Coli

Jim Hu jimhu at tamu.edu
Mon Jul 3 19:03:28 EST 2000



Chris LaRosa wrote:

> > Can someone give a working protocol
> >
> > for PCRing a gene from E.Coli genome?
>
> > Anton.
>
> Step 1) Design good primers.
>
> Step 2) Design good primers.
>
> Step 3) Isolate genomic DNA according to myriad protocols available on
> web with minimal search.  Also see Red cookbook or blue cookbook.
>
> Step 4) Read the manual of your PCR machine and or Taq DNA polymerase
> provider... Possible purchased from DnAMP Inc. or others.  Gibco BRL
> gives a protocol with every tube of Taq polymerase, for instance.

For those who want someone else to do steps 1 and 2 for large numbers of
genes, primer sets can be purchased from Sigma-Genosys...just for the
ORFs.

Actually, getting PCR products from E. coli is pretty easy - for plasmids
we just pick a colony into water in an Eppi tube, boil it, spin, take a
SMALL amount of the sup and amplify.  If anything, using too much DNA can
cause problems.  We've also done this for looking at lambda lysogens, so
chromosomal loci should work too.

I'd avoid Taq since errors can be a problem.  Pfu, Vent, HotTub etc. tend
to do a better job.  In any case, sequence the product to make sure it
matches the genome sequence.

The original poster listed a bcm.tmc address.  There are plenty of people
there who can help.






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