1 reply to this topic

[Niels]

Hi there

I have been done some reading in here, but found that there was not really any information on the inclusion of a melt curve step before moving on to sequencing.

Thus my plan is to:

1: Identify several promoters of interest (that I think are regulated by methylation by a specific chemical that I am interested in)
2: Design primers for BSP
3: Do the bisulfite treatment and
4: Run qPCR followed by a melting curve
5: in case of differences in melting curves between a control group (e.g. tissue from 6 animals) and a treatment groups (n=6) move on to sequencing. And in case no difference is found in melting curves skip the sequencing.

Does anyone have a comment on this approach, my wife says sequencing is not that difficult so perhaps just move directly to it without bothering about a melting curve. But on the other hand skipping a promoter and concentrate on others could save me valuable time and resources. Should I trust my wife on this one :-),  ?

Bw Niels

[pcrman]

I think your wife is right. Direct sequencing is not a big deal. I just don't know how sensitive melt curve method is for telling apart the difference of a few basepairs. BSP primers are prone to give primer dimmers which further interfere with melting property of PCR products. .