Reasons for PCR failure....?
Gregory S. Buzard 'Buz'
buzardg at MAIL.NCIFCRF.GOV
Wed Aug 7 16:00:02 EST 1996
On 2 Aug 1996, Bill Burnett wrote:
Okay, we've all been there.... One day the PCR's are great,
So what's the weirdest, wackiest reason you've ever come up
with for them not working?////
Well, so far I have seen people report bad water,
mysteriosly aged primers at -20, autoclave residue in
tubes, UV damaged oil, bad Mg++ or bad buffer (we have had
both), loss of cycling calibration on the machine, and bad
Let's see, we had a post-doc who's busy fingers reprogramed
our step-cycling files to ramping files, then there was the
bigger is better campaign, where if it didn't work just add
more template (the template had the inhibtor, so things
just got worse!), the leave out the Mg, 'hell, it's just
PCR'study, and the 'let's order these new cheaper tubes,
but not tell Buz' (they were thick-walled tubes). My
favorite all time wall-of-shamer was the different colored
(thin-walled) tubes that I thought would help keep track of
different parts of the experiment. Turned out that some
colors worked, some didn't, done in duplicates. Someone
else beat me to publishing that one.
When all else fails, go to some one's lab, sit at their
bench, use there reagents, get a successful reaction, get
your confidence back up, and dive back in. Or just go
fishing, that's what I do.
More information about the Methods