semi-specific PCR

Bryan L. Ford fordb at bcc.orst.edu
Wed Feb 25 14:40:24 EST 1998


Dom Spinella wrote:
> 
> Andrew Bahn writes:
> > Dear netters,
> >
> > I'm working on first strand cDNA-libraries that were immobilized on
> > magnetic beads. I try to amplify a "native" well known clone out of one of
> > these libraries with one specific primer (universe primer) and an oligo
> > dT25 (reverse primer). I tried different alternatives: 1. two rounds PCR,
> > then taking the supernatant for further PCR amplification; 2. 5 or 10
> > rounds PCR and then taking the supernatant for PCR; 3. PCR with the library
> > for 35 cycles; in every case a standard PCR protocol with an annealing of
> > 52-55=B0C was used. I always get a smear!! But if I test the specific revers=
> > e

> Andrew:
> 
> Undoubtedly, your smear contains your gene of interest in  addition to a lot of other
> products.  You could always try cloning the smear and probing the clones


Ho Ho, and it is always possible to tow a Mercedes-Benz with a team of
horses! 

If you have a probe then you have, or can easily have, the sequence
necessary to design specific PCR primer(s) to keep the "Mercedes" on the
road.


Sincerely,
Bryan L. Ford



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