sssI with genomic DNA - (Mar/07/2007 )
I´m new in bioforum. I´ve been working with comercial methylated DNA (chemicon), but now I´m starting new lab work on realtime methylation PCR and as I need loads of DNA to optimize my PCRs I´m thinking of methylating DNA myself.
I´ve been reading that some of you use sssI (from new england biolabs) and follow the protocol suggested by them.
Well, my questions are:
has someone tried this in genomic DNA?
do you perform one or two rounds of methylation to achive complete methylation of DNA?
do you use any purification kit afterwards?
Thanks a lot,
Thanks for your reply. If you don´t bother i have a few more questions:
Do you methylate full genomic DNA or do you methylate shorter regions?
Which columns do you use?
Thanks a lot,
what type of genomic DNA do you use?
if human, I tend to do two rounds of methylation. 37C overnight, then respiking the reaction with extra enzyme and SAM for another 4 hours @ 37C.
although in excess, if same depletes you don't get any more methylation.
@Nick - Didn't you get full methylation in one round? Just curious because my samples (human and rat) looked good.
@Ameli - I use DNeasy columns (which where mistakenly ordered twice) but with the DNA purification protocol from Qiagen QiAmp Kit (I only changed the elution volume). That works for me.
didn't check, but it doesn't hurt to do it twice to be sure to be sure.
Thanks for your replies.
I work with human DNA. But i´m doubting between:
- methylating full human genomic DNA or
- performing a first round of PCR (getting a bigger amplicon then my target region) and methylate this region and use this methylated region as a positive control for my Methylight experiments.
My doubts arise because I´m not sure if the methylation reaction would achive complete genomic methylation on human DNA.
there are pros and cons to your second method.
it would be easier to methylate an amplicon as opposed to gDNA.
But you must be vigilent and prevent carryover contamination that would lead you to a false positive.
another con for amplicons only:
if you want to test the efficafy of your measurements and control for bias it surely makes a difference if you PCR with amplicons or genomic DNA as templates.