How to detect incomplete enzyme digests - (Feb/05/2009 )
I'm performing enzyme digestions to detect methylation of the genome at certain locations and am worried that I'm getting an incomplete digest. I'm wondering what are some methods for detecting incomplete digestions?
controls, controls, controls.
well how about cutting samples of your DNA for progressively longer periods. 1hr, 2hr, ... overnight.
Would suggest you perform your digest as usual. Take sample after incubation and analyse (gel? or whatever). Then to the bulk reaction mix add a further aliquot of enzyme, incubate and reanalyse. If you get the same result from both reactions then primary digestion should be ok.
If you are worried about the presence of inhibitors in your sample, it can be useful to compare runs with different amounts of starting sample ( if your analysis method permits.
Hope this helps.