Daniel Gene Sinclair
danielg at earl.med.unc.edu
Wed May 30 08:55:59 EST 1990
In article <9005291727.AA13840 at genbank.bio.net> MBY134 at sysh.surrey.ac.uk writes:
> PCR REPRODUCIBILITY
>We are presently having difficulty with day to day
>reproducibility of the PCR. One day it works fine, the next,
>with the same reagents, it seems to either not work at all or
>the sensitivity is down 100-fold. We are using the HYBAID
>THERMAL REACTOR, Taq polymerase from GIBCO-BRL, frozen stocks
>of primers made on a Applied Biosystems synthesizer, buffer
>(with W1 detergent) and a standard dilute DNA prep.
>Has anyone had similar problems?
I was going to answer privately, but this will be fun (for me at least) to
discuss openly. The hybaid cycler is the one with no top on it, right? I
think that I would have stayed away from it in the first place, perhaps
heating is not good. Have you hooked up an xy plotter to your machine to
check the accuracy (sp?) of temperature control (no overshoot, etc.)?
Many thermal cyclers (TC's) have a output for this.
I imagine that the enzyme is ok, although until we get *our* pcr working
like clockwork, we're sticking with Amplitaq, even if it is expensive.
Our main source of trouble was with the primers. IMHO, you have to watch
for 'non-homogenous dissolution', i.e. the primers are sitting in the
bottom of the tube, not as a ppt, but perhaps in a, uh, gel-like state.
Ever have problems getting genomic dna into solution? Many times you get a
gel-like blob that doesn't go into solution even when you add more bffer
and heat at 50 degrees C (or so). These smaller (admittedly, much
smaller) pieces may behave similarly. I actually did an expt where I was
careful to *not* shake the primers after thawing, and I took from the top,
then took the same primers, mixed them by shaking, and took some for pcr,
and the first yeilded *nothing*, while the second gave the expected band.
Ok, so I'm not publishing yet, but it's worth considering. Also, we have
had our primers just *poop out* as we say, over time, perhaps from
repeated freeze thawing (aliquotting is the soln). When in doubt, make
your primers again and *this* time, take better care of them :-).
I'm unfamiliar with w1 detergent, what is it? I haven't made any buffer
for a while, and have forgotten what goes in it.
One additional note. If your sample dna is not in solution evenly, you
may just be adding buffer w/o dna in it. Of course, this is a long shot,
since pcr is pretty sensitive (our primers give an interpretable band down
to 0.1 ug genomic dna), but when you're troubleshooting, no idea is too
Fresh from PCR Land,
**** The shallow man has *** | Do not be overly righteous, nor overly
**** has opinions; *** | wise: why should you destroy yourself ?
**** the deep, *** | - King Solomon, wisest man of his
**** convictions. *** | day, Ecclesiastes 7:16.
More information about the Methods