Richard Lab:Restriction Digest protocol

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Revision as of 22:01, 20 October 2009 by Vaishnavi Ananth (talk | contribs) (New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="prepared DNA">prepared DNA <i><br><tab><div style="margin-right: 600px;">(from Miniprep, PCR or Gel Extraction)</div></i><...)
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Solutions/reagents:

  • <a name="prepared DNA">prepared DNA
    <tab>
    (from Miniprep, PCR or Gel Extraction)
    </a>
  • restriction endonucleases
  • 10X restriction endonuclease buffer
  • <a name="BSA">BSA
    <tab>
    (optional)
    </a>
  • phosphatase
  • phosphatase buffer
  • distilled water

Equipment:

  • Incubator
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning.
  2. Measure out <a href="#prepared DNA" >prepared DNA</a> 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):

    <thead></thead><tbody></body>
     10X restriction endonuclease bufferBSADNA solutionrestriction endonucleases
    Restriction Digestion<b>5 µl<b>1 µl<b>40 µl<b>1 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.
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