3 replies to this topic

[joyphyl]

Hello!

I got a problem with direct sequencing of BSP product.

When I sequenced the product with antisense primer, main peaks represent the right targeted sequence,
but problem is that peaks mostly appear as mixed nt. Especially G's are almost everywhere.

Would the sequencing with sense primer again help to get a better result?

[zlx0814]

joyphyl, on Jul 14 2009, 07:20 PM, said:

Hello!

I got a problem with direct sequencing of BSP product.

When I sequenced the product with antisense primer, main peaks represent the right targeted sequence,
but problem is that peaks mostly appear as mixed nt. Especially G's are almost everywhere.

Would the sequencing with sense primer again help to get a better result?

i am not think so ,since BSP product is a pool of many PCR products from many templets,including non-complete transition templets. you'd better cloning them into Vector ,and then sequencing.
good luck!

[pcrman]

If your PCR product is very pure, no matter there is methylation or not, you should obtain very nice chromatograph. Your problem is likely caused by interference from non-specific amplfication. How did the band look like, sharp and strong? How did you purify the DNA, Did you use the right amount of template and primer? Try to find a reliable sequencing service provider.

Sense primer usually only give worse results than reverse primer.

[joyphyl]

pcrman, on Jul 19 2009, 03:57 PM, said:

If your PCR product is very pure, no matter there is methylation or not, you should obtain very nice chromatograph. Your problem is likely caused by interference from non-specific amplfication. How did the band look like, sharp and strong? How did you purify the DNA, Did you use the right amount of template and primer? Try to find a reliable sequencing service provider.

Sense primer usually only give worse results than reverse primer.

Thank you for replying!


I purified the DNA using phenol-chloroform extraction, and bisulfite-treated with CpGenome modification kit from Chemicon.

For BSP, I used 30 ng of bisulfite-treated DNA as template in total 15 uL reaction.

I measured the concentration of bisulfite-treated DNA using nanodrop.

The BSP product band in gel looked quite sharp and strong, but the sequencing result was a mixed product.



Could too much template cause the problem?

,or incomplete bisulfite conversion could make this problem?

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