Human genomic restriction digests are used in the lab principally in generating
Southern transfers, i.e., screening, parent, and family blots. Most of the digests
are now prepared using the Biomek workstation. When only a few samples are
needed, it may be faster to prepare them manually, instead of writing a new
method for the Biomek.
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 mg 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 blot in our lab uses 4 µg of genomic DNA per gel
lane. We prepare digests for a minimum of 2 blots, and always include an
additional mg of DNA for the test digest, (therefore the minimum amount of
DNA to be digested for Southerns is 9 mg, 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/µL. The digests are prepared with the human
DNA at a concentration of 167 µg/µL.
2–3 days total time.
For each digest use (scale up accordingly):
36 µL human DNA (9 µg)
18 µL enzyme cocktail
54 µL total volume.
- Enzyme cocktail: Prepare about 10% more of the enzyme cocktail than
required, to allow for pipetting error, losses, etc.
To prepare cocktail for one 9-µg digest:
(scale up depending on the total number of digests), 5.4 µL 10X specific
restriction enzyme buffer 18–45 units of enzyme (units = 2 – 5 × the
number of micrograms), bring volume to 18 µL with sterile dH2O.
- Add 18 µL of the cocktail to 36 mL DNA in a labeled eppendorf tube. Mix
by tapping the tube with your finger. Quick-spin to remove bubbles.
- Prepare the test digest: remove 6 µL (= 1 µg human DNA) to another
labeled tube containing 1 µg (1–2 µL) lambda DNA. Mix and quick-spin
- Prepare one control digest for each restriction enzyme used: combine 2 µL
enzyme cocktail and 1mg lambda DNA in an eppendorf tube. Bring the
volume to 8 mL using sterile dH2O
- Incubate both sets of digests in the same incubator at the appropriate
temperature for 8 hours to overnight.
- Place the human-only digests in a –20°;C freezer (store until the results of
the test digest is known).
- Add 1 µL 10X glycerol dye mix to the test and control digests and run the
samples on an agarose gel. Also load a BRL 1-kb ladder marker lane. Run
the gel until the dyes are separated at least 1 inch. Stain and photograph.
- Add another 3-5 units enzyme/µg DNA (= 24-40 µ) to the incomplete
digests (be careful to keep the total amount of enzyme added less than
1/10 total digest volume). Incubate for another 6 hours to overnight. If the
test digest appeared less than 80% complete, also prepare another test and
control digest (otherwise, assume the extra enzyme and incubation will
- Add 5 µL 10X glycerol stop mix to each of the complete digests, mix, and
quick-spin. Store digests in the –20°;C freezer (good for a few months) or
in the –80°;C freezer for long-term storage.
- For 4 µg/lane (Southern gels), load 26 µL of the digest.