Restriction digestion

Virtualab Protocols (Chang Bioscience)
1. Digest 0.2-1 mg of DNA per 10-20 ml reaction. Miniprep DNA from 5 ml culture usually is in the range of 1-10 mg. Use 1/30 to 1/10 per digestion for analyzing the restriction patterns.

2. For multiple digestions, round bottom 96-well plates can be used. Otherwise use eppendorf tubes.

3. For the selected restriction enzymes, find the most appropriate restrition digestion buffer. For double digestions, a buffer should be chosen such that both enzymes would work well.

4. Make a reaction mix:

DNA (0.2-1mg) 2 ml
10X restriction digestion buffer 1 ml
10X BSA (10 mg/ml) (optional) 1 ml
Enzyme or enzymes 1-10 units
ddH2O to a total of 10 2l

5. The amount of enzyme should not exceed 10% of the total reaction volume. Because enzymes usually are stored in 50% glycerol. Excess glycerol in the reaction mix may inteferr with enzyme activities.

6. For multiple reactions, make a master mix without DNA first. Aliquot to each tube or well and then add DNA last.

7. Spin briefly to collect all liquid down to the bottom of the tube. For 96-well plates, gentlly tap the plate on the bench, and seal the plate with parafilm.

8. Put reactions in 37 C waterbath or incubator for 0.5-2 hours.