Long PCR

AShiflett ashiflett at mbl.edu
Thu Mar 25 16:13:37 EST 2004


Hi!  I am attempting to amplify an approximately 12 kb piece of DNA
that is near the telomere of a chromosome.  I have used both Herculase
from Promega and the Elongase enzyme from Invitrogen.  In both cases,
I do not obtain a product of the expected size.  I followed the mfg
directions, and performed optimization for primers, annealing temp,
and Mg concentration.  I use approximately 100 ng of genomic DNA which
is not sheared and looks great on a gel.  Does anyone have any tips
for amplifying long pieces or DNA, or can recommend a good polymerase
that has been successful for difficult PCRs?  Any advice is welcome
and appreciated.



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