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Methylation sensitive enzymes


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#1 CpGIsland

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Posted 16 February 2012 - 11:25 PM

Hi all,

So I have recently started experimenting with methylation sensitive res enzymes (prior have been using bisulfite to study methylation).

The enzymes (HpaII+HhaI) that i am using are methylation sensitive (they do not cut when CpG is methylated).

They were supplied by NEB.

Does anyone have experience of the efficiency of these enzymes to fully digest unmehtylated DNA?

I have been able to get them to cut 99% of the unmethylated DNA but there always seems to be a few copies left after digestion that I can detect using PCR primers that flank the restriction sites!

I have been mixing the tow enzymes together in a common buffer (NEB buffer 1) and I know that HhaI works better in NEB buffer 4.

Just wondering if anyones has accomplished complete digestion with methylation sensitive enzymes and can suggest a protocol?

Cheers

#2 methylnick

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Posted 29 March 2012 - 02:02 PM

have you tried spiking the reaction with more enzyme after incubation? i.e.: like having two rounds of digestion?




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