3 replies to this topic

[VULCANICA]

Hi,

are there any control primers which amplify bisulfite converted gDNA (Rattus).

[pcrman]

There are many types of controls, I am not sure what control you need. In general, you may include unmethylated and methylated DNA to include in bisulfite conversion and use the converted DNA as negative and positive control template in your PCR reaction, and amplify the DNA using primers that bind to region of interest.

[VULCANICA]

Hi PCRman,

I converted gDNA of a Rattus cell line by EZ DNA Methylation-Gold. I haven't achivied to amplify region of interest, yet. But I don't know whether primer pair work or converted DNA is adequate for PCR. Thus, I need a primer pair which conversion-specific and isn't contain CpG.

If there is a primer pair that working for bisulfite converted DNA amplification, could you send me sequence of primers and pcr conditions?

[pcrman]

I don't have validated rat BSP primers myself, but you can find some in the literature. You can use the tested primers to amplify your converted DNA.

Yes, you are right, when you have modified your DNA and tested your BSP primers without seeing amplification, and you want to figure out what is wrong. Problem could occur at multiple steps: such as loss of DNA, PCR problem, poor primer design, etc. But I would say the most problematic is PCR which needs careful optimization. You can find tips on that in this forum.