PCR failing

Sébastien Vigneau via methods%40net.bio.net (by sebastien.vigneau from gmail.com)
Tue Apr 6 09:48:18 EST 2010


Hi,

If the PCR used to work but is not working anymore, you may have some PCR
inhibitors in your samples. Diluting them in water, about 100 times, can
help (but trying different dilution factors, e.g. 10, 100 and 500 may be a
good idea). Alternatively, your template concentration may be too low or
your DNA may be degraded.

If this is a new PCR, you may lower the annealing temperature or increase
the concentration of primers (generally in the 0.2 µM - 0.8 µM range for the
final concentration). However, in the first case, you may loose specificity
and in the second, you may generate primer dimers.

I hope this helps.

Sébastien

On Tue, Apr 6, 2010 at 9:05 AM, Christian Praetorius <prae from gmx.net> wrote:

> LeboMad <lmadubanya from gmail.com> wrote:
>
> >have done all troubleshooting there is out there I can't seem to get
> >it right. I normally get nice clear bands but for some reason I get
> >very faint bands if any at all. Can anyone suggest anything. I've got
> >a student that needs some PCR runs and needs result as in yesterday.
>
> The nyou should describing *exactly* what your procedure is and what
> you tried to troubleshoot.
>
> Christian
>
> --
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-- 
Sébastien Vigneau

Lab: Marisa Bartolomei's laboratory - 360 Clinical Research Building - 415
Curie Blvd- Philadelphia - PA 19104-6148 - USA -- phone: +1-215-898-9277 --
fax: +1-215-573-6434


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