help! Long PCR

jim jim
Wed Jan 4 06:03:40 EST 1995


Help!!!

Does anyone out there have any tips on successful long PCR. I am trying
to PCR fragments of Mitochondrial DNA about 13kb in length using a mix 
of taqs (promega and taqextender).
What I consistantly get is either non specific banding or a pattern 
similar to partially sheared DNA. Hot starts and variations in cycle
parameters such as extension and denaturating time seem to have little
effect.
Are these patterns representative of a particular problem?
Does anyone have any advice on long PCR in general?
The thing that puzzles me is that I have already PCR'd 8kb so why does an
increase in several kb make things more complicated?
I have considered redesigning primers with a higher annealing temperature 
so as to make a two step PCR (denature and extension)but this sort of 
thing is only really cited as useful for really long PCR (20kb+) would it
really make such a difference?

Thanks for your time
Jim






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