snyder.9 at osu.edu
Mon Apr 29 23:04:27 EST 1996
In article <4m8nma$gg3 at taurus.fccc.edu>, "Warren D. Kruger"
<wd_kruger at fccc.edu> wrote:
> In my lab we have been trying to consistently amplify a 1.8 kb PCR
> product from 1st strand cDNA derived from lymphocytes. We use a nested
> PCR strategy. After the 1st 25 cycles, we take 1ul of the product and
> amplify another 25 cycles. Sometimes it works great, most of the time
> it doesn't. When it doesn't work we usually see a smear running through
> to the top of the gel. Occasionally we see our band within the smaear.
> Sometimes we see nothing at all. We have tried increasing the dilution
> of the 1st PCR, but this usually does not seem to help. We are using
> Kletaq/vent polymerase sold by Clonetech. Our control PCR reactions
> amplifying 1pg of cloned cDNA works well even after a single round of
> PCR. The template is moderately GC rich, (62%).
> What is the cause of the smear? How can it be so huge? Any clever
> ideas on how we can increase the "stability" of our reaction so it is
> consistant every time? My tech is going nuts.
are you sure that your primers aren't hitting in some conserved area,
(that they don't have enough homology to some other sequence(s) that if
they hit enough in the first few cycles the long product generated sucks
the primers away from your intended target? That might give you a smear
(from all that long nonspecific product) a good percentage of the time,
but a nice band every once in a while. Just an idea.
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