Method: Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
4/19/1990
C. Helms
Principle:
The following is a generalized example of a restriction digest.
Estimate the amount of DNA needed in your digest and scale up
accordingly. To visualize a digest on an ethidium bromide-stained
agarose gel, you will need to take the size of the fragments and the
total size of the clone DNA into account (e. g. 10-50 ng of intact
lambda-sized genomes (~50 kb) are easily seen on gels but if cut into
small (~1kb fragments), the relative proportion of the clone DNA in
each fragment is ~ 1/50 and more DNA (500-1000 ng) should be loaded
in order to see them).
If preparing a large number of digests at a time and the DNAs are at
the same concentration, prepare a cocktail of the reaction mix then
divide it among the tubes of DNA.
Time required:
Procedure:
A general rule of thumb is to use 1 µg clone DNA/ 10 µl in the final
digest reaction mix (Maniatis recommends 1 µg/20 µl).
Reaction Mix:
| 1.0 µl | 10 X enzyme buffer |
| 0.5 µl | 2-3 units restriction enzyme |
| 1-8.5 µl | 1 µg DNA (determine concentration on a gel before
setting up digest) |
| bring volume to 10 µl with sterile dH2O |
- 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.
- Quickspin to remove bubbles (DNA will adhere to bubble surface and
becomes inaccessible to the enzyme). Incubate at the recommended
temperature for 1 hour.
- Stop the reaction by adding 2.5 µl 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 µl 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.
