ANCHORED PCR

txpljfg at UABCVSR.CVSR.UAB.EDU txpljfg at UABCVSR.CVSR.UAB.EDU
Mon Apr 25 15:50:18 EST 1994


I have been attempting to use anchored PCR to clone T cell receptor
beta chain transcripts from small tissue samples.  However, we have
been having trouble obtaining sufficient product for cloneing. 
Examination of the ethidium bromide stained gels shows no bands, but a
southern blot of the PCR products using an internal primer indicates
bands of the appropriate size.

We have optimized the reaction conditions for Mg, annealling temp,
extension times.  We have tried using DMSO (from 5-15%), but get bettwe
results without it.  We are tailing the cDNA with poly C and using an
anchor primer with a poly-G sequence.

Is there an important parameter that I have missed?

Any hints?

Thanks Loads in advance.

==============================================================================
James F. George, Ph.D.              "Back off man, I'm a scientist"
Department of Surgery                --Bill Murray
University of Alabama at Birmingham
205-934-4261 voice
txpljfg at uabcvsr.cvsr.uab.edu
===============================================================================



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