how many colonies should I send to sequence? - (Nov/28/2005 )

Hi, all
After bisulfite treatent and PCR, I subclone the PCR products into TOPO vector. Then, I pick two plasmids for sequencing after checking them by digestion (the size of them lookscorrect). The results of sequencing showed that the methylation status of CpGs are different between the two plasmids. Now, I have some questions:
1. why are there different methylation status from the same PCR product after TOPO cloning?
2. Why do people usually send 15-20 plasmids to sequence for one PCR product?
3. Will COBRA give me some clues if I don't want to sequence so many colonies?

Thanks a lot

Qiong

1. why are there different methylation status from the same PCR product after TOPO cloning?
A: there are 3 possibilities. 1st is that two alleles are differently methylated; the 2nd is that individual cells are differently methylated; the 3rd is the combination of 1st+2nd.

2. Why do people usually send 15-20 plasmids to sequence for one PCR product?
A: people think 10-20 clones can well represent the actual methylation status. I compared the methylation percentage from 10 clones with direct sequencing result and found the two correlate pretty well.

3. Will COBRA give me some clues if I don't want to sequence so many colonies?
A: I think so.

Hope that helps.