simple protocol for colony PCR

Anthony Palombella palomb at beagle.Colorado.EDU
Tue Oct 18 21:17:17 EST 1994


Robert.Coelen at jcu.edu.au writes:

>Hi,

>Could anyone give me a simple protocol for direct PCR of plasmid DNA from 
>bacterial colonies, taken directly of the plate. The emphasis is on simple !
>I need such a protocol for work in a developing nation.


I have successfully PCR'd from both coli genomic DNA and plasmid DNA by
picking a colony from a plate with a sterile toothpick or P-20 tip and
resuspending the colony in a standard PCR reaction mix.  In my hands, it
often takes two rounds of amplification:  I see little product after 30
cycles or so, but using ca. 5 ul of that reaction in a new reaction gives
me good results.  In the first reaction expect to see chromosomal DNA and
RNA on the gel.  It takes very few cells for this to work, much less than
an entire colony.  Good luck!

				-- Tony
-- 
>>>>>>>>>>>>>>>>>>><<<<<<<<<<<<<<<<<<<<
>>Ehh... I could be wrong, ya know...<<
>>palomb at beagle.colorado.edu         <<
>>>>>>>>>>>>>>>>>>><<<<<<<<<<<<<<<<<<<<



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