PCR of a long genomic fragment... HELP?

Thu Mar 18 15:32:05 EST 1993

(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:
   200ng genomic DNA; boiled
   2.5 pmol each primer; 30mers with 60% GC content
   200mM dNTPs
   1 nmol G32 protein

   80C - 'hot start' add 5 U Taq
Then, 30 cycles of:
   94C - 30"
   69C - 30"  (72degrees also works well)
   72C - 2'30" + 1" per cycle; 72 - 5' after last cycle

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 am interested to have any hints and ideas for PCR of long fragments.  
I am currently making first strand cDNA to try and work out the conditions 
for the primers before extending the system to the genomic fragment.

Thanks in advance for your sage advice,

Paul Waterhouse, Ph.D.
Ontario Cancer Institute
Toronto, Ontario

pwater at galen.oci.utoronto.ca

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