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 &mu;L reaction. 100 &mu;L reactions are also common especially if your DNA to be cut is dilute.''


 * 5 &mu;L NEB2 buffer (for all digests with BioBricks enzymes, we use NEB2 buffer. It keeps things simple and seems to work).
 * X &mu;L DNA (usually ~500 ng depending on downstream uses).
 * 0.5 &mu;L 100X BSA (added to all digests because BSA never hurts a restriction digest)
 * 1 &mu;L BioBricks enzyme 1 (regardless of the volume of the reaction, 1 &mu;L 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 &mu;L of enzyme since it is sticky due to the glycerol content.)
 * 1 &mu;L BioBricks enzyme 2
 * (42.5 - X) &mu;L 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 &mu;L 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 &mu;L, 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.
 * 7) 4-6 hour incubation at 37&deg;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.
 * 8) 20 mins at 80&deg;C to heat inactivate enzyme. This step is sufficient to inactivate even Pst I.
 * 9) 4&deg;C forever (or until you pull the reaction out of the thermal cycler).
 * 10) Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction.