Genomic bacterial DNA PCR.

[Koen De Smet]

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:

either:
		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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