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 (~1 kb 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.
A general rule of thumb is to use 1 mg clone DNA/ 10 mL in the final digest
reaction mix (recommendation 1 µg/20 µL).
- 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°;C or add 0.5 µL of 0.5 M EDTA.