Reasons for PCR failure....?

Bruce Amsden bamsdsen at prozyme.com
Fri Nov 1 20:13:14 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.  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.
>
>
You don't say whether just one pair of primers aren't working or if
all your reactions are a bust.  If nothing works, including whatever
positive control you have, then you might try new dNTPs.  I can't say
I've ever seen them go off "overnight", but I did have one instance
where they were the culprit.

Bruce Amsden



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