capillary PCR

Mr V. Schoenfeld DOM vs10005 at crc.ac.uk
Thu Apr 21 13:14:20 EST 1994


Some netters asked me for more details about PCR in capillary tubes,
so here we go:
-the tubes are easy to load:you just dig them in your mix and capillary
action does the rest
-you can run between 5 and 30 ul reactions per tube
-nothing special about the conditions, except that you need to add BSA which
prevents the enzyme to coagulate against the glass
-you can add fycol to the reaction so that you can load your sample directly from
the glass tube in your agarose gel
-the ends of the tubes are sealed with a flame

The machine does not actually stop at denaturing and annealing temperatures,
it just gets there and carries on. It's fab!


Also, concerning the PCR labelling of hybridisation probes, I thought one
of the receipes I saw on the News was a bit funny. So here's mine:

3 ul 10 uM primer 1
3 ul """""""""""" 2
3 ul dNTP mix (2mM dATP, dGTP, dTTP and .2 mM dCTP)
3 ul 32P dCTP
3 ul 15 mM Mg buffer
0.5 ul TAQ
3ng plasmid DNA
water up to 30 ul

Go for 35 cycles, denature and add the lot to your hyb. solution. 

That's fine for a S.blot; Do two reactions for gridded libraries and three for plated ones.
(Mind you, you will need to vary the total volume depending on the number of recombinants 
you're screening, obviously).

Hope it helps!

Vince
-------------------------------------------------------------

Vincent Schoenfeld
University Department of Medicine
Addenbrooke's Hospital
Cambridge CB2 2QQ, UK

vs10005 at med.cam.ac.uk          | Phone: (0223)402436/336853
vs10005 at crc.ac.uk              | Fax:   (0223)21136
 





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