hubahopp at gmx.de
Sun Mar 28 16:55:49 EST 2004
You'll have to fiddle with lots of parameters, probably, especially when
you have a difficult template. To reduce misprimings (which will of course
increase with the langth of the template), there are polymerase
preparations on the market that are optimized for that (see on of my last
As easiest approach, you might try a gradient-touch down PCR, preferably in
a gradient cycler, with say 12 tubes in parallel. Be sure that the
extension time is long enough, depending on the polymerase (Taq 5-7 kB/min,
Pfu, Pwo approx 1 kb/min, before you optimize for the single band of
desire, be sure that you can get it).
You may check your primers by setting up PCRs with internal primers having
a shorter distance (500 - 1000 b from the ends, if the positions of the
primers in your gene are ABCD, so run PCRs AB and CD).
Can you assess the quality of your DNA by amplifying anything else?
Consider to do a long melting step of 10 min at 96degC and a stop on
ice-water before you add polymerase and maybe nucleotides.
At 13:13 25.03.2004 -0800, AShiflett wrote:
>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
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