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rogier stugerNOstSPAM at cellbiology.uni-frankfurt.de.invalid
Thu Jan 20 13:49:47 EST 2000

A very fast and easy method to check for insert is to do a PCR directly
on your colonies. No need to miniprep, no need for restriction enzymes.
All you need are 2 primers (which may be universal primers found in
every lab).

the protocol:
use a yellow tip to pick up a colony (make sure to take the entire
colony. if you also pick up some agar, that's no problem).
transfer the colony to 100 ul sterile water, and pipet up and down
until you get an even suspension.
Use 3 ul of this suspension in a standard 20 or 25 ul PCR reaction:
start with a 5 min. 95 C incubation to release the DNA from the cells.

Primer suggestions: best thing is a primer that bites your insert and a
primer that sticks to your vector.
You can also take two vector-specific primers. For most commercial
cloning vectors, up- and downstream sequencing primers are OK.

Good luck,
Dept MicFizz, Free U of A
Amsterdam, the Netherlands
E rogier at biogate.com

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