Reasons for PCR failure....?
affouj at rpi.edu
Fri Aug 2 14:24:29 EST 1996
with regards to your request for reasons we've come up with
for pcr failure....
[This ended up being a bit long, so sorry! skip it if it's boring....]
Reasons for PCR failure:
1) Vinnie wasn't used...explanation: we have an older MJR cycler,
and the latch that holds the (unheated) lid closed broke a while
ago. so, to keep it closed we place a little plastic statuette of
a man with arms outstretched with a heart around his neck that says
'i love you this much!' (which the lab tech found in the woods
behind his house one day) unfortunately, vinnie was found sans nose
anda foot thanks to a pack of wild dogs...anyway, the point is that
our lab was moved elsewhere on campus, and due to the new location
of the cycler, there isn't enough room above the cycler for vinnie
to stand, so we now use a nice bottle of triton-x to weigh the lid
down. we believe vinnie is the key to pcr success (see #2)
2) The proper donation to Vinnie's scholarship fund was not made. we
used to have a little box in front of the thermocycler labelled
'vinnie's college fund,' which you were supposed to put coins in
before you turned on the thermocycler so that vinnie would be a
kind, benevolent deity rather than a mean and nasty one.
a bit more rational reasons???
3) the mineral oil and the water...these seem to be blamed for EVERYTHING,
but since the mineral oil can't be UV-treated (an article a while
back about how this was bad, but followed up by another which spoke of
repairing UV damage...but anway) we like to blame the oil :) i think
we've done eveything possible to water to treat it, and get it from
as many different sources as possible.
4) you tried TOO hard to be aspetic/sterile. often i find that if pcr
hasn't been working properly, use reverse psychology - DON'T use
autoclaved pcr tubes, tips, ANYTHING :) this essentially guarantees
the success of the reactions...hehehe
5) we have found that the age of the primers is critical - not even talking
about freeze/thaw effects, since we aliquot them out. after they've
been in the -20 for a while (months) they seem to decrease in efficiency...
6) gene-32? well, it depends on the day, really...some times it seems to
help reduce inhibition/increase amplification, some times it doesn't. yet
another contributor to what we like to call 'the mysteries of pcr.'
7) and finally, remember what pcr really stands for:
PRIMERS COMBINE RANDOMLY :) so matter what you do, it won't help!
good luck to all, and i hope i didn't go on TOO long here, but it's just
been one of those days (trying out a new oligo in my PCRs...which of course
brings me to my final axiom: "If you are trying something for the first time,
it will work wonderfully. After that, kiss your good luck goodbye."
affouj at rpi.edu
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