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Method: Restriction Digestion of Plasmid, Cosmid, and Phage DNAs

4/19/1990

C. Helms


Principle:

Time required:

Procedure:

Reaction Mix:

1.0 l10 X enzyme buffer
0.5 l2-3 units restriction enzyme
1-8.5 l1 g DNA (determine concentration on a gel before setting up digest)
bring volume to 10 l with sterile dH2O

  1. Put the water and buffer into the tube first, then add the enzyme (avoid putting enzyme into water first, as it may start to break down). Put the DNA in last and mix by tapping the tube with your finger.
  2. Quickspin to remove bubbles (DNA will adhere to bubble surface and becomes inaccessible to the enzyme). Incubate at the recommended temperature for 1 hour.
  3. Stop the reaction by adding 2.5 l 5 X Ficoll dye mix if the sample is to be loaded directly onto a gel; otherwise stop the reaction by placing it at -20 degrees C or add 0.5 l of 0.5 M EDTA.

References:

Sambrook, J., Fritsch, E.F., and T. Maniatis.(1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. pp 5.28 - 5.32.