PCR from single colonies?

[Ian J. Mehr]

In article <4da4q0$fma at mark.ucdavis.edu>, 
nelange at ucdavis.edu says...
>
>Does anyone have a good protocol or advice for doing PCR 
directly from 
>single bacterial colonies?
>
>thanks in advance,
>
>Nathan
>

Nathan,

For PCR off chromosomal DNA we use a solution of TE and 
0.5% Triton X-100.  To 100ul of this cell lysis solution 
(CLS) we add a piece of filter paper which was used to 
pick a colony off an agar plate.  This collects more 
consistent amounts of colony than a toothpick or loop, 
which becomes important when comparisons between PCRs off 
colonies are necessary.  The filter paper brand is moot, 
and we cut ours to small strips of about 3mm x 5mm.  The 
CLS and filter paper are vortexed for 1', heated to 95oC 
for 15', and vortexed for 1'.  We then use 2 to 5ul of 
this mix in a standard 30 cycle PCR reaction, which works 
great.  We store the remaining CLS+DNA at -80oC till we 
need it again, and I've used the DNA in this manner for 
several months with many freeze/thaws with no adverse 
effects.  

Alternatively, one can maintain a viable culture of the 
colony picked by first placing the filter paper with 
bacteria into 100ul of freezing media containing glycerol, 
vortexing, and taking 50ul to freeze at -80oC, and 50ul to 
add to 50ul of a 2x stock of CLS to use in PCR.  This way, 
one can simply access the viable frozen stock to grow the 
strain of interest should the PCR experiment turn out 
desirable.

Hope this helps,

Ian Mehr
Grad Student
Northwestern University
Microbiology-Immunology
ijm at nwu.edu