Standard DNA single or double digest of variable volume V using NEB restriction enzymes.
- X μL DNA template
- NEB Digestion Buffer # (# = 1, 2, 3, or 4)
- 10mg/ml BSA
- Determine compatibility and reaction conditions using Double Digest Finder (double digest only).
- Determine reaction volume V by computing V = [(X + 1)/0.8] μL (single digest) or V = [(X + 2)/0.8] μL (double digest).
- To DNA sample, add
- 0.1V 10X Buffer Y
- 0.1V 10mg/ml BSA
- 1 μL restriction enzyme 1
- 1 μL restriction enzyme 2 (double digest only)
- Incubate 3 hr to overnight at reaction temperature given by NEB (standard = 37 °C).
- Optional: Heat inactivate restriction enzyme(s) by incubation 80 °C 20 min. Not necessary when digestion product is going to be purified or gel extracted, as these procedures remove the enzymes.
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- SC 14:15, 29 June 2012 (EDT):