NEED PROTOCOL OF MINIPREP - URGENT

Jürgen Häußler juergen.haeussler at medizin.uni-ulm.de
Tue Jan 25 16:07:38 EST 2000


Dear Rogier
Just put the tip in the already prepared PCR-reaction for I few seconds, remove
it  and do the cycles like you do it everytime. This will save additional time
Jürgen

rogier schrieb:

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