Quick PCR

Andre Hamel hamel at ccu.umanitoba.ca
Thu Apr 22 18:03:36 EST 1993


I simply place a LITTLE from a colony (touch a colony, DON'T gouge into
the colony so much that there's readilly visible amount on toothpick),
resuspend this into 50 uL water. Place into thermocycler PRESET to 95oC,
let sit in there for few minutes (5 usually enough), vortex, quick ufuge
at top speed, then transfer as much as 30 uL from top (avoid "pellet" ..
it's there at bottom of tube) into standard 100 uL PCR rx'n mix.
 
Start off the PCR rx'n with a 3 min soak at 95oC ... that's all             
^^^^^ ^^^ ^^^ ^^^ ^^^^ ^^^^ ^ ^ ^^^ ^^^^ ^^ ^^^^                  

I've used this routinely for confirming a clone's insert size (using
universal F. and R. M13 sequencing primers (50 ng each) ... 30 to 40
cycles suffices ... also used this for PCR with degenerate primers (8 to
32 fold degen.), except used 500 ng each primer.

 regards
Andre Hamel
Manitoba Vet Virol
Winnipeg, Manitoba
Canada

email: hamel at ccu.umanitoba.ca


In article <116464 at bu.edu> pfoster at bu.edu (Patricia Foster) writes:
>Netters,
>I have been told that it is possible to toothpick an
>E. coli colony and go almost directly into PCR.
>Anyone have a protocol?
>
>Pat Foster
>Boston University School of Public Health
>Boston, MA
>pfoster at med-itvax1.bu.edu
>



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