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Materials

Digest Mix

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

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.