Richard Lab:Restriction Digest protocol

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(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><...)
Current revision (02:44, 20 November 2009) (view source)
 
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<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></a></li><li>restriction endonucleases</li><li>10X restriction endonuclease buffer</li><li> <a name="BSA">BSA <i><br><tab><div style="margin-right: 600px;">(optional)</div></i></a></li><li>phosphatase</li><li>phosphatase buffer</li><li>distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><font color = "red"><i>Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning.</i></font><br></li></p><p><li>Measure out <a href="#prepared DNA" ><font color=#357EC7>prepared DNA</font></a> into sterile 1.5-ml microcentrifuge tube (1).<br>Add  <b><font color=#357EC7>4</font></b> volumes <font color=#357EC7>distilled water</font>.<br>Use the following table as a checklist for preparing the reaction in sterile 1.5-ml microcentrifuge tube (2):<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>10X restriction endonuclease buffer</font></td><td><font color=#357EC7>BSA</font></td><td><font color=#357EC7>DNA solution</font></td><td><font color=#357EC7>restriction endonucleases</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Restriction Digestion</font></td><td><b><b><font color=#357EC7>5 µl</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td><td><b><b><font color=#357EC7>40 µl</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td></tr></body></table></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for at least <b><font color=#357EC7>1 hr</font></b>.<br><font color = "#800517"><i>Use a water bath for incubation.</i></font><br></li></p><p><li>Perform enzyme inactivation by storing at <b><font color=#357EC7>75°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li><b><font size=2>If digesting vector:</font></b><br>Measure out <b><font color=#357EC7>1 µl</font></b> of <font color=#357EC7>phosphatase</font> into sterile 1.5-ml microcentrifuge tube (2).<br>Add <b><font color=#357EC7>5 µl</font></b> of <font color=#357EC7>phosphatase buffer</font>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for at least <b><font color=#357EC7>45 mins</font></b>.<br>Perform enzyme inactivation by storing at <b><font color=#357EC7>75°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li>Store at <b><font color=#357EC7>-20°C</font></b>.<br></li></p></ol></html>
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<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></a></li><li>restriction endonucleases</li><li>10X restriction endonuclease buffer</li><li> <a name="BSA">BSA <i><br><tab><div style="margin-right: 600px;">(optional)</div></i></a></li><li>phosphatase</li><li>phosphatase buffer</li><li>distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><font color = "red"><i>Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning.</i></font><br></li></p><p><li>Measure out <a href="#prepared DNA" ><font color=#357EC7>prepared DNA</font></a> into sterile 1.5-ml microcentrifuge tube (1).<br>Add  <b><font color=#357EC7>4</font></b> volumes <font color=#357EC7>distilled water</font>.<br>Use the following table as a checklist for preparing the reaction in sterile 1.5-ml microcentrifuge tube (2):<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>10X restriction endonuclease buffer</font></td><td><font color=#357EC7>BSA</font></td><td><font color=#357EC7>DNA solution</font></td><td><font color=#357EC7>restriction endonucleases</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Restriction Digestion</font></td><td><b><b><font color=#357EC7>5 µl</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td><td><b><b><font color=#357EC7>40 µl</font></b></td><td><b><b><font color=#357EC7>4 µl</font></b></td></tr></body></table></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for at least <b><font color=#357EC7>1 hr</font></b>.<br><font color = "#800517"><i>Use a water bath for incubation.</i></font><br></li></p><p><li>Perform enzyme inactivation by storing at <b><font color=#357EC7>75°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li><b><font size=3>If digesting vector:</font></b><br>Measure out <b><font color=#357EC7>1 µl</font></b> of <font color=#357EC7>phosphatase</font> into sterile 1.5-ml microcentrifuge tube (2).<br>Add <b><font color=#357EC7>5 µl</font></b> of <font color=#357EC7>phosphatase buffer</font>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for at least <b><font color=#357EC7>45 mins</font></b>.<br>Perform enzyme inactivation by storing at <b><font color=#357EC7>75°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li>Store at <b><font color=#357EC7>-20°C</font></b>.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 2 hrs, 15 mins</font></b></p></html>

Current revision

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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