PCR primer problem ?

EK someone at microsoft.net
Sun Jan 18 01:19:48 EST 2004


"Helge Lind" <helgelind at yahoo.com> wrote in message
news:b0d5bc33.0401130714.2a1c70d2 at posting.google.com...
> I am currently having some problems with my PCR and I think it could
> have something to do with one of my primers. The primer is quite long
> (43 bp) and was accidentaly left in room temperature for about a week.
>
> Does anyone know a method for checking my primers for degradation ?
> Denaturating gel electrophoresis ?
>
> Best regards
>
> Helge

Boil your primers for 3-5 min and quickly cool down in ice to assure no or
very low level of self dimers or hairpins and run them in an 8%
polyactilamide gel. Same rigs you use for protein minigels would be OK.
Detect with ethidium bromide or SIBR green. Also, you could try
reprecipitating your oligo or repurifying using miniprep columns (check the
ones that work with ssDNA of your length), as well as try to add more of the
primer to the PCR reaction to see if this results in appearence of the
desired product.
Emir





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