Reasons for PCR failure....?

Eitan Rubin bcrubin at dapsas1.weizmann.ac.il
Thu Aug 8 03:51:12 EST 1996


This definitely falls under "too much sterility (see first posts in this thread).
  Eitan Rubin.

Mayumi Yagi 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.
> 
>  Hope this helps,
> Mayumi



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