Reasons for PCR failure....?

George Rutherford gruther at bilbo.bio.purdue.edu
Wed Aug 7 11:15:52 EST 1996


In article
<Pine.A32.3.92a.960805092007.18304A-100000 at homer25.u.washington.edu>,
Mayumi Yagi <myagi at u.washington.edu> wrote:

> Hi, Bill,
> 
> The one that took me the longest time to figure out had to do with a
> residue left in the tubes after autoclaving.  Apparently the autoclave was
> malfunctioning, leaving water (and some mysterious unknown stuff,
> presumably from the water pipes or something) in the tubes, and the
> student helper was simply leaving them in an incubator to dry afterwards.
> So, depending on how much water was left, I would get tubes from the same
> container that would work fine, and others that totally killed the PCR.
> It was especially fun because at the time I was trying to optimize several
> different primer pairs, and it got to the point where I couldn't get
> duplicates set up at the same time and run on the same machine to give me
> the same results.  Finally I realized that the tubes didn't seem to be
> wetting evenly (crooked meniscus).  Once we got the autoclave fixed,
> things seem to have settled down to more manageable levels.
> 

Try DMSO or glycerol (not in combination). These sometimes help. SSB
sometimes also can help, although rarely in my experience. Try running
widdershins about the machine three times with your right hand on your
head.

George
>  Hope this helps,
> Mayumi
> 
> On 2 Aug 1996, Bill Burnett wrote:
> 
> >
> > Hi gang...
> >
> > Okay, we've all been there....  One day the PCR's are great, next day
> > they don't work.  You change everything, they still don't work.  You
> > clean, polish and sterilise everything and they still don't work.  You
> > try multiple different water sources, you try different batches of taq,
> > you prepare them on ice, you prepare them at RT, you do hot start, you
> > don't do hot start, you check your cycler, you don't wear aftershave,
> > you make new templates, you use old templates that used to look great...
> > And they still 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*.
> >
> > Help!!!
> >
> > B.
> >
> > ----------------------------------------------------------------------------
> > Bill Burnett: bbur at dml.ac.uk
> > Scottish Association for Marine Science
> > P.O. Box 3, Oban, Argyll, PA34 4AD.
> >
> >
> >
> >
> 
> ------------------------------------------------------------------------------
> Mayumi Yagi, Ph.D.
> Research (151)
> Veterans Affairs Medical Center
> 1660 South Columbian Way
> Seattle, WA 98108
> Tel (206)762-1010, ext 1918
> Fax (206)764-2598



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