Wondering if you could help me again.
I'm trying to analyse the methylation state of my transgenic promoter in my rice plants and there are two copies of the transgene in the rice line that I'm working on. I've bisulfite-treated my DNA, cloned the PCR fragments and picked 10 colonies for sequencing. So I am wondering:
1) I've been trying to find out why you pick multiple clones and it seems to be because of incomplete conversion. Is this correct? If so then:
2) In my initial results there is sequence variation between the different clones but as these are clones from the same bisulfite-treated DNA and the same PCR can I assume that any differences are due to the differences in the methylation state of the two promoters? Unfortunately (despite a number of attempts) we haven't been able to find out where the two promoters are inserted into the rice genome, so I have no way of distinguising between them.
Thanks in advance,