2 replies to this topic

[annat]

Hello,

I'm planning to perform a bisulfite treatment

1.I'm working with Rat DNA. What negative and positive controls can I use? There are'nt any commercially available control kits with Rat methylated/non-methylated DNA (as far as I understand)..What should I do and how can I check my experiment?

2. I'm using MethPrimer to built the primers for my experiment. It's finding 3 CpG islands (with many CpG nucleotides inside) at the promoter region of my gene of interest. Is it OK to check only one specific area with ~ 15 CpG inside in order to determine that the "promoter of the gene X is methylated/unmethylated"? Or maybe I should check every single CpG found at the promoter region?

3. What materials should I use for the PCR amplification of converted DNA? some specific polymerase? maybe you can recommend me some kit/mix that can be easily added to the sample?

Thanks a lot!!! I would really appreciate any help/suggestions!
Anna

[pcrman]

Hi Anna,

1. Control DNA is not a must. The purpose of using control DNA (methylated and unmethylated) is to make sure that  bisulfite conversion is complete and your primers are good. If you don't have controls, that's fine.

2. If you find multiple CGI in the promoter sequence, you can first focus the one close to the transcription start site because methylation of this region has a greater impact on transcription.  

3.  For PCR, I would suggest that you obtain some hotstart taq polymerase. I highly recommend JumpStart RedTaq polymerase from Sigma. Other components should be the same as regular PCR.

Good luck!

[annat]

pcrman, on Jun 18 2009, 03:07 PM, said:

Hi Anna,

1. Control DNA is not a must. The purpose of using control DNA (methylated and unmethylated) is to make sure that  bisulfite conversion is complete and your primers are good. If you don't have controls, that's fine.

2. If you find multiple CGI in the promoter sequence, you can first focus the one close to the transcription start site because methylation of this region has a greater impact on transcription.  

3.  For PCR, I would suggest that you obtain some hotstart taq polymerase. I highly recommend JumpStart RedTaq polymerase from Sigma. Other components should be the same as regular PCR.

Good luck!

Thanks a lot for you help!!!! You are great!