help! Long PCR

Ron Kagan rkagan at ewald.mbi.ucla.edu
Wed Jan 4 20:17:22 EST 1995


In article <D1vo24.JoL at info.swan.ac.uk> 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
>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?

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).

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"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)

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Ron Kagan
rkagan at ewald.mbi.ucla.edu



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