help! Long PCR

Ron Kagan rkagan at
Wed Jan 4 20:17:22 EST 1995

In article <D1vo24.JoL at> jim harvey,  writes:
>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
>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
>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?

Maybe you could try the Perkin-Elmer GeneAmp XL kit for long PCR.  They
advertise tht it can give products up to 35 kb.  (I haven't actually used
it myself, though).

"You cannot strengthen one by weakening another; and you cannot
 add to the stature of a dwarf by cutting the leg off a giant."

                                - Benjamin Franklin (1706-1790)

Ron Kagan
rkagan at

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