PCR which worked fine previously giving high molecular weight smear

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Murali
Murali's picture
PCR which worked fine previously giving high molecular weight smear

I run Reverse Transcription PCR. The PCR product is 240bp. I use 22mer primers.
Program
Intial denaturisation - 95deg C - 90 seconds
40 cycles
Denaturisation 95 deg C- 30 second
Anealing 64 Deg C - 30 second
Extension 68 Deg C- 1 minute
close loop
Final extension 68 Deg C-2 minutes

The PCR was working fine till couple of weaks. Now I get only a high molecular weight smear ( most DNA stuck on the well). I have changed following to check
1. Changed the primer stock
2. Reautoclaved all the materials used
3. Have tried with different cDNA samples which were working previously.
4. Used different PCR machines.
5. GAPDH house keeping gene gives a good product for the cDNA samples

All of them now giving a high molecular weight smear.Please advise.

IVF
IVF's picture
Just read this paper in

Just read this paper in pubmed