simple protocol for colony PCR

Ferenc Marincs agpfxm at pnv.palm.cri.nz
Tue Oct 25 19:46:43 EST 1994


In article <385bkr$opd at osiris.wu-wien.ac.at>,
Sebastian.Bunka at vu-wien.ac.at wrote:

> Robert.Coelen at jcu.edu.au wrote:
> : 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 didn't try it with plasmid dna, but for amplification of a genomic
> fragment:
> Grow overnight on (in my case blood agar), 
> strain Actinobacillus spec.; pick some colony material and dissolve
> in distilled water (OD660 0.1-0.25 about 10E8-9 bugs, boil for 10 minutes;
> use 2 microliter in 50 microl. of your PCR setup; for my gene I run
> the PCR with 25 cycles (40 sec. 94 dC, 40 sec. 50 dC, 90 sec. 72 dC, and
> a last cycle 5 min. 72 dC) when I run 10 microl. on a gel I have nice
> visible bands at 1700 bp).
> 
> Hope this helps, Sebastian
> --
> 
> email:                   [ Sebastian.Bunka at vu-wien.ac.at ]
> voice:                   FAX:
> +43-1-71155260           +43-1-7149110
> Location: earth, europe, austria, vienna  Inst. of Bacteriology  Vet.Univ.

I usually resuspend some colonies from the agar plate in 50 ul of water
and use 5 ul form that suspension in the 100 ul PCR reaction without any
treatment. 5 ul bacteria from an overnight cultured broth works also very
well in my hands.
Good luck, Frank

Ferenc Marincs
AgResearch, Private Bag 11008, Palmerston North, New Zealand
Phone: +64-6-356 8019, Fax: +64-6-351 8032
E-mail: agpfxm at pnv.palm.cri.nz



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