PCR

Ed Rybicki ED at micro.uct.ac.za
Tue Nov 24 05:47:13 EST 1992


> If anyone could possibly help with information pertaining to PCRing a
> 2000bp sequence, please do so by sending a Vax mail message to this
> address.  I have been attempting to amplify an IGS region from a
> Laccaria bicolor dik. collector Kropp using sequenced primers at
> approx. 15 b (depending on the primer).  I have used low annealing
> temperatures but have only once achieved a fragmented amplified area.
> If anyone has any tips or advice please respond.


First off, not wise to do too low an annealing temp as the primers might
well amp up non-specifically.  Second, you may want to include up to 10%
v/v DMSO, as this definitely helps amp up long templates (I could amp a
2.7 kb circular virus DNA in 2 pieces of 1.5 and 1.3 kb only when I used
DMSO, even with Cetus AmpliTaq).  Third, you may want to up your
denaturation temp a degree or so and keep it there for a full 60-90 sec
(94degC is good).

Hope is useful,

  ____________________________________________________________________
 | Ed Rybicki, PhD             |    "Now you've got the hang of it    |
 | (ed at micro.uct.ac.za)        | There's nothing you can't do with it |
 | Dept Microbiology           |        If you're very into it        |
 | University of Cape Town     |         You can't go wrong...."      |
 | Private Bag, Rondebosch     |                                      |
 | 7700, South Africa          |               -Mad John              |
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