I run Reverse Transcription PCR. The PCR product is 240bp. I use 22mer primers.
Intial denaturisation - 95deg C - 90 seconds
Denaturisation 95 deg C- 30 second
Anealing 64 Deg C - 30 second
Extension 68 Deg C- 1 minute
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.