Reasons for PCR failure....?

Jacalyn S. Newman jsd119 at email.psu.edu
Sat Nov 2 13:47:40 EST 1996



> Bill Burnett <bbur at dml.ac.uk> wrote:
 

> >Hi gang...

> >Okay, we've all been there....  One day the PCR's are great, next day
> >they don't work. .....
> >
> >So what's the weirdest, wackiest reason you've ever come up with for 
> >them not working?
> >
> >At this point, after 2 months, I'll try *anything*.


Boy is this familiar! On a bad day, I blame the "Gremlins." Seriously, I
found that the type of gloves I was wearing had an affect. When I was using
latex gloves, with all that powder, successful PCR was random at best. I
now use non-latex gloves that have cornstarch instead of the latex powder.
They are blue, but I can't think of the brand name off the top of my head.
Email me if you need the brand. When I started using the blue gloves, I
started getting PCR to work. It may be coincidence, but I suspect the latex
powder was getting into some of my tubes and killing the reaction.

I'd also be careful how many times your reagents are frozen and thawed. I
make small aliquots of my dNTP's, say 20µl, so I only freeze and thaw them
a couple of times before they are used up. I've read that Mg+2 can come out
of solution and also affect your PCR. You can heat your Mg+2  solution to
60° or so to make sure it's dissolved. I have the paper filed at work, so
if you need the reference, again, just email me.

When I run into failure now, I make sure that all of my reagents are fresh,
including the water. I use water purchased for tissue culture. It's
sterile, DNAse free, RNAse free, and pretty cheap. Using it also guarantees
that your water quality won't change from experiment to experiment.


When all else fails, remember PCR stands for Pure Chance Reaction. Keep
your sense of humor, and keep trying. You'll get it to work.

 I hope this helps.

Jacalyn S. Newman
jsd119 at email.psu.edu



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