Knight:Restriction Digest

Materials

• Restriction enzymes (EcoR I, Spe I, Xba I or Pst I) from NEB
• NEB2 buffer
• BSA
• Deionized, sterile H₂O

Digest Mix

Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.

• 5 μL NEB2 buffer (for all digests with BioBricks enzymes, we use NEB2 buffer. It keeps things simple and seems to work).
• X μL DNA (usually ~500 ng depending on downstream uses).
• 0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest)
• 1 μL BioBricks enzyme 1 (regardless of the volume of the reaction, 1 μL enzyme is used because generally this represents a 10-25 fold excess of enzyme and is therefore sufficient for most digests. Also, it can be difficult to accurately pipet less than 1 μL of enzyme since it is sticky due to the glycerol content.)
• 1 μL BioBricks enzyme 2
• (42.5 - X) μL deionized, sterile H₂O

Procedure

1. Add appropriate amount of deionized H₂O to sterile 0.6 mL tube
2. Add restriction enzyme buffer to the tube.
   Vortex buffer before pipetting to ensure that it is well-mixed.
3. Add BSA to the tube.
   Vortex BSA before pipetting to ensure that it is well-mixed.
4. Add appropriate amount of DNA to be cut to the tube.
   Vortex DNA before pipetting to ensure that it is well-mixed.
5. Add 1 μL of each enzyme.
   Vortex enzyme before pipetting to ensure that it is well-mixed.
   Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
6. Place in thermal cycler (MJ Research, PT-200) and run digest protocol.
   1. 4-6 hour incubation at 37°C
      Use a longer incubation time if you have time or are worried about the efficiency of cutting. I think this time can be shortened to 2 hrs while still cutting to completion.
   2. 20 mins at 80°C to heat inactivate enzyme.
      This step is sufficient to inactivate even Pst I.
   3. 4°C forever (or until you pull the reaction out of the thermal cycler).
7. Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction.