Double Digestion - double digestion for southern (May/03/2005 )
I want to do a double restriction enzyme digestion and then use the product for a southern blot.
My southern protocol calls for 10ug of gDNA. My enzymes are XbaI and PstI.
What would be the appropriate volumn for the double digestion, and what would be the correct amount of time for this digestion. Also what is the deal with BSA? Do I want it in this reaction?
Your volume depends on the 10 ug sample. Our lab rule is that a digest volume should be at least twice the DNA solution component. That being said, I suggest as small a volume as possible (maybe 20 ul), so concentrate your DNA.
Buffer - look at the double digest section of the NEB catalogue and it will tell you about the optimal buffers. If a single digest with EITHER enzyme requires BSA, add this to the double digest... so check out the product inserts that came with the enzymes. If in doubt, toss some in.
Time - another reason to look at the product insert is that it will tell you the rate of the two enzymes (usually I think 1 ug /hr ...ish). Sixteen hours should be enough. Make sure your enzymes don't have nuclease activity or 'star activity' (non-specific digestion) ... once again in the product insert.
Positive - digest DNA (ex lambda) with each and both of the enzymes to make sure they work under those conditions before investing your precious 10 ug of hard-pruified DNA.