Method: Manual Method of Restriction Digestion of Human DNA

June 24, 1990

C. Helms

Principle:

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 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.

Time required:

2- 3 days total time

Procedure:

Day 1

For each digest use ( scale up accordingly):

36 µl human DNA ( 9 µg) 18 µl enzyme cocktail ----------------- 54 µl total volume

1. Enzyme cocktail: Prepare about 10% more of the ezyme 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 X the # µgs)
   bring volume to 18 µl with sterile dH2O

2. Add 18 µl of the cocktail to 36 µl DNA in a labeled eppendorf tube. Mix by tapping the tube with your finger. Quick-spin to remove bubbles
3. 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 as before.
4. Prepare one control digest for each restriction enzyme used: combine 2 µl enzyme cocktail and 1µg lambda DNA in an eppendorf tube. Bring the volume to 8 µl using sterile dH2O
5. Incubate both sets of digests in the same incubator at the appropriate temperature for 8 hours to overnight.

Day 2

1. Place the human-only digests in a -20 degrees C freezer (store until the results of the test digest is known).
2. 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 1kb ladder marker lane. Run the gel until the dyes are separated at least 1 inch. Stain and photograph.
3. Add another 3-5 units enzyme/ µg DNA (= 24-40 u) 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 complete it).
4. Add 5 µl 10X glycerol stop mix to each of the complete digests, mix and quick-spin. Store digests in the -20 degrees C freezer (good for a few months) or in the -80 degrees C freezer for long-term storage.
5. For 4 µg/ lane (Southern gels), load 26 µl of the digest.

References: none