Genomic bacterial DNA PCR.

Koen De Smet k.desmet at
Fri Feb 20 03:33:51 EST 1998

Mr SCG Rison wrote:
> Hi,
> Has anybody got any protocols/suggestions on how to perform PCR with a genomic
> (E. coli) bacterial DNA template.  Is there a direct way or do I first have to
> isolate genomic DNA and then PCR (and if so, what protocol(s) do you suggest?).

There are several methods to do PCR without prior extraction of E.coli DNA that have in 
the past been discussed on this newsgroup. The one I tend to use is probably no better 
or worse than others, but it works in my hands so I stick with it:

		Scrape colony off plate and suspend in 50 -100 ul water
		Spin 50 ul culture down and suspend pellet in 50 -100 ul water
Spin bacteria down and suspend pellet in 50 -100 ul chloroform
Add equal volume of water
Vortex vigorously
Spin to separate phases
Use 1-5 ul of top (water) phase in a PCR reaction

Other methods involve adding the colony straight into a PCr reaction or boiling a colony 
for 5-10 minutes in water and using that in a PCR reaction.

Koen De Smet
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     Imperial College School of Medicine at St Mary's									

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