- SC 14:47, 19 June 2012 (EDT):
Standard DNA digest protocol (NEB restriction enzymes).
For a DNA digestion of total volume V (in μL):
- ~0.8V DNA
- 0.1V 10X digestion buffer (1, 2, 3, or 4; NEB)
- 0.1V 10mg/ml BSA
- 1 μL restriction enzyme 1
- 1 μL restriction enzyme 2 (optional, for double digestion)
- Find appropriate enzymes(s), associated buffer, reaction temperature from .
- Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.
- Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
- Add the reaction components together in order listed above. Mix gently and spin.
- Incubate at reaction temperature for 3 hr to overnight.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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Relevant papers and books
- Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773
or instead, discuss this protocol.