Team:Brown/Notebook Protocols/redigest

 DNA digestion protocol & hints 

Overview: Although it is pretty standard to digest DNA with restriction enzymes, here is a standardized protocol and some hints…

Materials:

• DNA sample in water or TE buffer

• 10x digestion buffer

• restriction enzyme

• DNA loading buffer

• Agarose gel 0.8% (or different depending on expected band sizes)

Procedure:

1. Pipet the following into a microfuge tube:

20 µl reaction 50 µl reaction DNA 0.1 to 4 µg 0.1 to 4 µg 10x Digestion buffer 2 µl 5 µl Enzyme (as appropriate) Water (Rest of volume)

2. Add the enzyme (1-5u/µg DNA)

3. Incubate at recommended temperature for an hour.

4. Take 2 to 5 µl of the digested sample, add loading buffer, and run on agarose gel to check the result.

Tips:

1. DNA:

• For checking DNA, use 0.1 µg; (or 4-8 µl from a good DNA mini prep)

• For cloning, 4 µg DNA is enough

2. Buffer: besides the buffer that comes with the enzyme, buffers from other company can be used, too (as long as the contents are the same)

3. Enzyme: the maximum volume that an enzyme can be used is 1/10 of the total reaction volume (example: 2 µl for 10 µl reaction)

4. Incubation time: can be longer than 1 hr, but don’t do overnight digestion if you don’t have to (genomic DNA requires overnight digestion)

5. Gel: make sure to run the uncut DNA along with the digested DNA. And, always run a DNA marker!

Reference: Current protocols in molecular biology (3.1.1 - 3.1.2)