Enzyme digestion before bisulfite treatment - (Jan/09/2005 )
Does enzyme digestion before bisulfite treatment make a big difference - i am having a few problems with incomplete conversion.
If so, can anyone reccomend me a good 'general' enzyme to use - I know it is probably best to select an ezyme that cuts nearest the area I am looking at, but my current study is one of several that will be perfomed on the DNA - so other regions may not have the same optimal enzyme. Is there one that cuts at fairly regular intervals across the genome and will be good for most future studies - any reccomendations would be gratefully recieved!
When selecting an enzyme, what parameters would you suggest - what is the optimal flanking distance between the cut site and region i want to sequence, and does it matter what sort of cutter I use (blunt-end vs one that leaves an overhang).
If you are going to study multiple genes, using restriction enzyme to reduce DNA size may be not a good idea unless you can find one or more than one enzyme that won't cut within your target regions.
Another simple method of reducing DNA size is mechanical shearing by passing DNA through a narrow gauge needle for a couple of times.