DNA denaturing problem for bisulfite sequencing - (Nov/29/2004 )

Hey,

I have another question:

In the protocol, it suggests that use RE to digest genomic DNA to give short fragments of DNA. This will give better denature results. My question is generally how long the DNA after RE digestion will give the good result ? In my case, I can find RE which only give me 5kb fragment after digestion. Is it good enough?

Thanks,
jimmy

Hi jimmy,

Although there is no study which defines the optimal restriction fragment size for bisulfite treatment, it seems to me 5kb may not make a big difference from intact DNA. I think you can use any enzyme (4-6 cutter) that won't cut within your target sequences. Alternatively, you can just shear your DNA by passing through a narrow gauge needle for a couple of times.