PCR-sequencing on colonies

ajwatson ajwatson at romeo.caltech.edu
Thu Mar 31 18:38:47 EST 1994


In article <CnJBn9.6w8 at biobase.aau.dk>
kjaer at biobase.aau.dk (Svend Kjaer) writes:

> I would like to hear from anybody, who has succesfully done PCR-sequencing
> directly on bacteriacolonies grown on plates. If anyone could advice me
> some literature or practical approaches I would be very thankfull.

I do this routinely.  Simply touch a toohpick to the colony, resuspend
it 50
ul of dH2O, boil it for 2-5 min, microfuge out the debris, and use
about 5 ul
or so in a 50 ul reaction.  USe the sequencing primers that flank the
insert.  PCR amplify a control plasmid (no insert) as well.  One of my
labmates claims to done it simply by taking part of the resusupended
plasmid (no boiling) -- but I don't know about this first-hand.

Cheers,

John



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