PCR of supercoils
Frank O. Fackelmayer
Frank at Fackelmayer.de
Sun Jul 29 12:40:19 EST 2001
"Dr. Duncan Clark" wrote:
>> But you are not really comparing like for like if I follow correctly.
> When you run a PCR out on a gel it is probably always after it has
> reached plateau in the PCR so you wouldn't expect to see much in the way
> of a difference in product yield even if you had a 10-100 fold copy
> number difference at the start (obviously depending upon how many cycle
> you are running but assuming that most people actually use too many).
> With real time you will see the same rough yield at plateau but when you
> are at quantification you are looking at when the PCR product is still
> amplifying exponentially i.e. very early in the PCR. At that stage you
> would see any differences in starting copy numbers that may be due to
> differences in supercoiling.
I agree. My understanding of the original post was, however, that the
poster implied there would be a different yield AFTER a "standard" PCR
with (I agree again) too many cycles, and that you only had to add some
"magic" stuff (that the poster may want to commercialize) to improve the
yield of PCRs from supercoiled plasmids. That is what I doubt.
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