Methylation studies - (Sep/20/2013 )

Hi!

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.

What kind of samples are you working on: DNA from cultured cells, tissues, etc, how big is the product size, at which step the problem occurs (no product, cloning issues), how do your PCR conditions look like?

The two issues you listed are not common causes behind BSP failure. 

I work with 3D samples that consists of cells encapsulated inside a self-assembling peptide. 

My product is 321bp and there is no problem in the amplification step (5min-94ºC + 40 cycles (45s-94ºC 45s-58ºC 90s-72ºC) + 10min-72ºC. Ramps of heating and cooling work at 2,5ºC/min). Normally my problems appear during cloning and more specifically during the ligation step. I use TOPOTA cloning kit from invitrogen and normally I obtain colonies but all of them contain the autoligation of the vector. For this reason I think that it is possible that the fragment to introduce in the vector have a secondary structure.