How to create a fully methylated control? - (Mar/12/2013 )
Most people are using the methyltranferase M.SssI to creative fully methylated controls. However, from what I have read, M.SssI methylates only CpG sites. Thus, the rest of Cs of the genome which are not followed by a G remain unmethylated (or have random methylation status depending on the sample). So, this is not a fully methylated control.
Do you know any way to have a truly completely methylated control, independently from the species?
Thanks a lot,
You buy one from Qiagen (human). The prize is worth the effort.
Thanks for the answer.
I'm not working with human and I need to have a DNA from my species (2 different fish). Any other idea?
You can PCR with 5-me dCTP instead of dCTP in the reaction. You'll have to mix with dATP, dGTP, and dTTP yourself.
Hi, most methylated DNAs from commercial parties are not entirely methylated.
A friendly lab once tried with replacing the Cytosine base in dNTP with 5-methylcytosine and doing a whole genome amplification reaction.
the are still using it and generally happy.
Also an excelent way to make fully unmethyled DNA, do a whole genome amplification using normal cytosine when making your dNTP mixture.
the kit they used was officially for human but it worked for their insect species. maybe order a sample at a supplyer and give it a go?