PCR Cloning from cDNA libraries

docon at midir.ucd.ie docon at midir.ucd.ie
Fri May 19 10:27:28 EST 1995


If anyone has experience of the following problems, then the benefit of
their experience would be greatly appreciated!
The problems include the amplification of what seems originally to be
the correct band(-1300bp) using sequence specific primers, which upon
sequencing are identified as cloning vector derivatives. This problem
has surfaced over a range of "optimized" conditions.
Secondly, fragments putatively identified by southern hybridisation are
proving refractive to nested PCR. This also has been carried over a
range of PCR conditions and is as yet unresolved. The only idea I have
is that the 5' region is extremely GC rich and perhaps this is
affecting annealing of the nPCR 5' primer.
Any suggestions please?
dave
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