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Warren watchcity at my-deja.com
Thu Jan 20 20:46:56 EST 2000

Hello, First 1 question:  Do you have any control to determine if the
restriction enzymes are working?  Choose a known vector or some other plasmid
you have a lot of (that you would expect to be cleaved), and test the
enzyme(s) for the expected result.  If you have 2 enzymes, testing each
respectively for linearization should also do the trick.  Of course you
should also include DNA size standards of the correct range.

In article <16e1e28c.f02ca681 at usw-ex0101-008.remarq.com>,
  rogier <stugerNOstSPAM at cellbiology.uni-frankfurt.de.invalid> wrote:
> 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).

SNIP A variation on the PCR technique uses a relatively dry selective medium
agar plate.  Grid the plate, and mark pcr tubes with the same  designation. 
Don't forget to date the plate, and explain what the vector, insert, and
selective marker used is!

> 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).

SNIP Use a filter tip if possible. After getting the colony, gently touch the
tip onto a grid area a few times.  Wrap the plate well to avoid drying out,
and transfer to 37C.

> 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.

SNIP If you wish, you can transfer the colony to a small (50ul) amount of
liquid selective medium instead.  Grow 37C until the liquid is starting to be
cloudy.  Mix the culture carfully to break up clumps, and take 1 ul of
culture per 10ul of PCR final.	Same PCR conditions.  The saved liquid
culture can be used to start on ON mini-prep, after confirmation of insert
and before the plate grows dense enough to harvest.

> 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

Good luck, and do all the controls!  Warren

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