Hello Jim,
The site http://www.BioProtocol.com is a good one. Give it a try.
Best of luck.
Davinderpal S Bhatia
Jim Hu wrote:
> 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.
