I have been working to set a protocol for E-cadherin methylation status during the last year. I have set the PCR conditions and protocol after bisulfit conversion (Methylcode-Invitrogen) and the conditions to clone the fragment obtained with TOPO TA cloning kit.
Well, my problem is that the protocol set sometimes work and sometimes not. Right now and after study different factors I have some teories but I am not sure if it is possible.
- Aliquoted primers can be degraded after a while if they have been always at -20ºC?
- Is it possible that the amplified sequence present a secondary structure which do not allow a correct ligation? How can I solve this issue? I have tried to decrease the ramp temperatures and at the end of the PCR the temperature is decreased slowly.
Thanks you very much in advance for your help.