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:
Hi gang...
Okay, we've all been there.... One day the PCR's are great, 
next day...

/Snip..../

So what's the weirdest, wackiest reason you've ever come up 
with for them not working?////

Bill,

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 
Karma.

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.

Buz 





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