Digestion-PDF

Overview

Standard DNA single or double digest of variable volume V using NEB restriction enzymes.

Materials

• X μL DNA template
• 10X NEB Digestion Buffer Y (Y = 1, 2, 3, or 4)
• 10mg/ml BSA

Procedure

1. Determine compatibility and reaction conditions using Double Digest Finder (double digest only).
2. Determine reaction volume V by computing V = [(X + 1)/0.8] μL (single digest) or V = [(X + 2)/0.8] μL (double digest).
3. To DNA sample, add
   • 0.1V 10X NEB Buffer Y (Y = 1, 2, 3, or 4)
   • 0.1V 10mg/ml BSA
   • 1 μL restriction enzyme 1
   • 1 μL restriction enzyme 2 (double digest only)
4. Incubate 3 hr to overnight at reaction temperature given by NEB (standard = 37 °C).
5. 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.

• Lab standard double digestion uses X = 22 μL plasmid DNA, so V = (22 + 2)/0.8 μL = 30 μL.
  • 22 μL plasmid DNA
  • 3 μL 10X NEB Buffer Y
  • 3 μL 10mg/ml BSA
  • 1 μL restriction enzyme 1
  • 1 μL restriction enzyme 2