PCR of a long genomic fragment... HELP?
finney at AMBER.MGH.HARVARD.EDU
Mon Mar 22 00:31:56 EST 1993
In article <930318153205.20800551 at HIPPO.OCI.UTORONTO.CA>, PWATER at HIPPO.OCI.UTORONTO.CA writes:
>(I hope that this isn't too much of a "PCR FAQ", but...)
>I have been trying to PCR a 3.5 kb fragment of genomic DNA using primers
>derived from exon sequence, with little success. An adjacent 3.0 kb fragment
>was amplified under the same conditions
>For the 3.5 kb fragment I have tried:
>* Mg++ at 2, 3, 4, 5 and 6 mM
>* annealing at 65, 67, 69, 71 and 73 degrees C
>* Taq, Vent and Pfu polymerases
>* the primers are 30mers with 60% GC content
>* combinations with 2 5' and 2 3'primers
>* nested primers; 30 cycles with outside then
> 20 or 30 cycles with inside primers
>* I get a smear with 40 cycles, but no specific band, even on a Southern
I and my friends have had some success with single-strand binding proteins.
Both T4 gene 32 protein and E. coli SSB work. These are heat-stable
proteins whose normal function is to increase the processivity of DNA
polymerases by inhibiting secondary structure in single-stranded DNA.
They are available from several sources. For greatest effect, they
should be added to roughly equal amounts (by weight) to the amount
of DNA you expect to be produced by the reaction. The main
drawback is that this can be expensive if you do it often.
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