Reasons for PCR failure....?

[Sailesh Surapureddi]

Hi

        Here some of the reasons that I picked up for PCR, which might be
helpful to you.

1. Clean template DNA (preferably Qiagen purified) @ low conc in sterile water
   or TE.

2. DEPC free water,, after long struggling.. I have found that Autoclaved
Milli
   Q water to be the best.

3.  Re calibrate your pipettes. (if the calibration is screwed up, you
might be
    adding excess of primers etc etc and there goes your PCR to dogs). In
your
    case, I guess this is the problem, since you have written everything
except
    about the pipettes

4. Powder free gloves (if you happen to wear any, most of the  times the powder
   from the gloves tend to inhibit reactions).

5. Avoiding the formation of bubbles while pipetting (a common mistake, usually
   neglected).

Last but not the least TONS of GOOD LUCK (..just kidding)

Sailesh.