Hiya,
now that there's 10 colony-PCR protocols in this thread, here's another
foolproof one:
pipet 100 ul sterile water into a tube. your pipet tip is now still
sterile, of course. use this tip to pick up the entire colony (doesn't
matter if you pick up some agar too), resuspend the colony into the
water.
Use 3 ul of this suspension for a standard 25 ul-PCR reaction. the
remaining 97 ul can be stored in the fridge for at least 2 months. use
20 ul of the suspension to start a 25-ml culture of scoring clones, so
you can make tons of DNA and never have to prep that plasmid again.
if you want to check your constructs by sequencing, just sequence the
PCR product. no need to do a miniprep. make sure to remove the PCR
primers first by spinning through a QiaQuick column.
Want to PCR on yeast? put clone in PCR buffer, boil for 10', add
polymerase and go cycle.
Good luck,
Rogier
Rogier Stuger
Dept MicFizz, Free U of A
rogier at biogate.com
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