Richard Lab:Restriction Digest protocol

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Solutions/reagents:

Equipment:

  • Incubator
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning.
  2. Measure out prepared DNA into sterile 1.5-ml microcentrifuge tube (1).
    Add 4 volumes distilled water.
    Use the following table as a checklist for preparing the reaction in sterile 1.5-ml microcentrifuge tube (2):

     10X restriction endonuclease bufferBSADNA solutionrestriction endonucleases
    Restriction Digestion5 µl1 µl40 µl4 µl
  3. Incubate at 37°C for at least 1 hr.
    Use a water bath for incubation.
  4. Perform enzyme inactivation by storing at 75°C for 15 mins.
  5. If digesting vector:
    Measure out 1 µl of phosphatase into sterile 1.5-ml microcentrifuge tube (2).
    Add 5 µl of phosphatase buffer.
    Incubate at 37°C for at least 45 mins.
    Perform enzyme inactivation by storing at 75°C for 15 mins.
  6. Store at -20°C.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 2 hrs, 15 mins

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