June 24, 1990
Because of the complexity of the human genomic DNA (~ 3 billion bp), any restriction digest can be expected to produce what appears to be a continuum of fragment sizes, with no individual fragment bands visible. It is impossible to tell from the ethidium bromide stained pattern whether or not the DNA digest is complete or only partially complete. To help determine the extent of digestion, we set up a test digest. The test digest is prepared after the human genomic digest is set up: an aliquot of the human digest is combined with 1 µg lambda DNA (lambda being a relatively simple genome of ~48 kb in size). The complete digestion of the lambda genome produces a distinct banding pattern on a gel superimposed upon the faint smear of the human genomic DNA. We assume that if the lambda DNA had been digested to completion, the human DNA digest must also be complete. After the incubation period,the human-only digests are stored in the freezer until the result of the test digests is known.
The standard Southern blots in our lab uses 4 µg of genomic DNA per gel lane.We prepare digests for a minimum of two blots, and always include an additional µg of DNA for the test digest, (therefore the minimum amount of DNA to be digested for Southerns is 9 µg, and the following procedure is based on this amount). The human DNAs used in the lab are adjusted to a concentration of 200-250 µg/ml. The digests are prepared with the human DNA at a concentration of 167 µg/µl.
For each digest use ( scale up accordingly):
36 µl human DNA ( 9 µg) 18 µl enzyme cocktail ----------------- 54 µl total volume
To prepare cocktail for one 9 µg digest:
5.4 µl 10X specific restriction enzyme buffer
18-45 units of enzyme (units = 2 - 5 X the # µgs)
bring volume to 18 µl with sterile dH2O