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Method: Manual Method of Restriction Digestion of Human DNA

June 24, 1990

C. Helms


Principle:

Time required:

Procedure:

Day 1

For each digest use ( scale up accordingly):

  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 g digest:

    5.4 l 10X specific restriction enzyme buffer
    18-45 units of enzyme (units = 2 - 5 X the # gs)
    bring volume to 18 l with sterile dH2O

  2. Add 18 l of the cocktail to 36 l 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 l (= 1 g human DNA) to another labeled tube containing 1 g (1-2 l) lambda DNA. Mix and quick-spin as before.
  4. Prepare one control digest for each restriction enzyme used: combine 2 l enzyme cocktail and 1g lambda DNA in an eppendorf tube. Bring the volume to 8 l 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 l 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/ g 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 l 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 g/ lane (Southern gels), load 26 l of the digest.

References: none