DNA extraction - Salting Out protocol

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(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="Digestion Buffer">Digestion Buffer <i><br><tab><div style="margin-right: 600px;">(10mM NaCl, 10mM TRIS (pH 8.0), 10mM EDTA...)
Current revision (02:08, 20 November 2009) (view source)
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="Digestion Buffer">Digestion Buffer <i><br><tab><div style="margin-right: 600px;">(10mM NaCl, 10mM TRIS (pH 8.0), 10mM EDTA (pH 8.0), 0.5% SDS)</div></i></a></li><li> <a name="Proteinase K">Proteinase K <i><br><tab><div style="margin-right: 600px;">(20mg/ml)</div></i></a></li><li> <a name="Sodium Acetate pH 5.2">Sodium Acetate pH 5.2 <i><br><tab><div style="margin-right: 600px;">(3M)</div></i></a></li><li>ice-cold 98% ethanol</li><li>ice-cold 70% ethanol</li><li>1X TE</li><li>water</li><li>tissue</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Tissue Digestion</font></b><br><ol type="a"><p><li>Measure out <a href="#Digestion Buffer" ><font color=#357EC7>Digestion Buffer</font></a> into a sterile 1.5-ml microcentrifuge tube.<br>Add <b><font color=#357EC7>0.005</font></b> volume <a href="#Proteinase K" ><font color=#357EC7>Proteinase K</font></a>.<br><font color = "#800517"><i>That is, for each ml of Digestion Buffer, add 5 µl of ProteinaseK.</i></font><br></li></p><p><li>Homogenize tissue in solution.<br></li></p><p><li>Incubate at <b><font color=#357EC7>55°C</font></b> for <b><font color=#357EC7>1 - 12 hrs(overnight)</font></b>.<br></li></p><p><li>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7>4°C</font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into a sterile 1.5-ml microcentrifuge tube.<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Precipitation of Protein and Cell Debris</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>0.1</font></b> volume <a href="#Sodium Acetate pH 5.2" ><font color=#357EC7>Sodium Acetate pH 5.2</font></a>.<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Incubate at <b><font color=#357EC7>-20°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>20 mins</font></b> at <b><font color=#357EC7>4°C</font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into a sterile 1.5-ml microcentrifuge tube.<br>Discard bottom layer.<br><font color = "#800517"><i>Be careful not to transfer any of the white solid (cell debris and SDS) into the fresh tube.</i></font><br></li></p></ol></li></p><p><li><b><font size=3>Precipitation of Nucleic Acids</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>ice-cold 98% ethanol</font>.<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Incubate at <b><font color=#357EC7>-20°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>20 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ice-cold 98% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ice-cold 70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p></ol></li></p><p><b><font size=3>(Optional)</font></b><br>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ice-cold 70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air. <br><p>Option 1: Add <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>1X TE</font>.<br>(or)<br>Option 2: Add <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>water</font>.<br></p><p>Resuspend the pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>Ensure to dry the pelletted DNA completely before attempting to resuspend.</i></font><br></li></p></ol></html>
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="Digestion Buffer">Digestion Buffer <i><br><tab><div style="margin-right: 600px;">(10mM NaCl, 10mM TRIS (pH 8.0), 10mM EDTA (pH 8.0), 0.5% SDS)</div></i></a></li><li> <a name="Proteinase K">Proteinase K <i><br><tab><div style="margin-right: 600px;">(20mg/ml)</div></i></a></li><li> <a name="Sodium Acetate pH 5.2">Sodium Acetate pH 5.2 <i><br><tab><div style="margin-right: 600px;">(3M)</div></i></a></li><li>ice-cold 98% ethanol</li><li>ice-cold 70% ethanol</li><li>1X TE</li><li>water</li><li>tissue</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Tissue Digestion</font></b><br><ol type="a"><p><li>Measure out <a href="#Digestion Buffer" ><font color=#357EC7>Digestion Buffer</font></a> into sterile 1.5-ml microcentrifuge tube (2).<br>Add <b><font color=#357EC7>0.005</font></b> volume <a href="#Proteinase K" ><font color=#357EC7>Proteinase K</font></a>.<br><font color = "#800517"><i>That is, for each ml of Digestion Buffer, add 5 µl of ProteinaseK.</i></font><br></li></p><p><li>Homogenize tissue in solution.<br></li></p><p><li>Incubate at <b><font color=#357EC7>55°C</font></b> for <b><font color=#357EC7>1 - 12 hrs(overnight)</font></b>.<br></li></p><p><li>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7>4°C</font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (3).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Precipitation of Protein and Cell Debris</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>0.1</font></b> volume <a href="#Sodium Acetate pH 5.2" ><font color=#357EC7>Sodium Acetate pH 5.2</font></a> to sterile 1.5-ml microcentrifuge tube (3).<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Incubate at <b><font color=#357EC7>-20°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>20 mins</font></b> at <b><font color=#357EC7>4°C</font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (4).<br>Discard bottom layer.<br><font color = "#800517"><i>Be careful not to transfer any of the white solid (cell debris and SDS) into the fresh tube.</i></font><br></li></p></ol></li></p><p><li><b><font size=3>Precipitation of Nucleic Acids</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>ice-cold 98% ethanol</font> to sterile 1.5-ml microcentrifuge tube (4).<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Incubate at <b><font color=#357EC7>-20°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>20 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ice-cold 98% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ice-cold 70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p></ol></li></p><p><b><font size=3>(Optional)</font></b><br>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ice-cold 70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air. <br><p><b>Option 1: </b>Add <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>1X TE</font>.<br>(or)<br><b>Option 2: </b>Add <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>water</font>.<br></p><p>Resuspend pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>Ensure to dry the pelletted DNA completely before attempting to resuspend.</i></font><br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 13 hrs, 27 mins</font></b></p></html>

Current revision

Solutions/reagents:

Equipment:

  • Incubator
  • Centrifuge
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Tissue Digestion

    1. Measure out Digestion Buffer into sterile 1.5-ml microcentrifuge tube (2).
      Add 0.005 volume Proteinase K.
      That is, for each ml of Digestion Buffer, add 5 µl of ProteinaseK.
    2. Homogenize tissue in solution.
    3. Incubate at 55°C for 1 - 12 hrs(overnight).
    4. Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 2 mins at 4°C and aspirate out the top layer.
      Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (3).
      Discard bottom layer.
  2. Precipitation of Protein and Cell Debris

    1. Add 0.1 volume Sodium Acetate pH 5.2 to sterile 1.5-ml microcentrifuge tube (3).
    2. Close the tube tightly and gently mix the contents by inverting the tube.
      Incubate at -20°C for 15 mins.
      Centrifuge at maximum speed for 20 mins at 4°C and aspirate out the top layer.
      Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (4).
      Discard bottom layer.
      Be careful not to transfer any of the white solid (cell debris and SDS) into the fresh tube.
  3. Precipitation of Nucleic Acids

    1. Add 2 volumes ice-cold 98% ethanol to sterile 1.5-ml microcentrifuge tube (4).
    2. Close the tube tightly and gently mix the contents by inverting the tube.
      Incubate at -20°C for 15 mins.
    3. Centrifuge at maximum speed for 20 mins at 4°C, gently aspirate out the supernatant and discard it.
    4. Add 1 ml of ice-cold 98% ethanol.
      Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 5 mins at 4°C, gently aspirate out the supernatant and discard it.
      Add 1 ml of ice-cold 70% ethanol.
      Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 5 mins at 4°C, gently aspirate out the supernatant and discard it.
  4. (Optional)
    Add 1 ml of ice-cold 70% ethanol.
    Vortex the mixture for a few secs.
    Centrifuge at maximum speed for 5 mins at 4°C, gently aspirate out the supernatant and discard it.

  5. Dry the pellet in air.

    Option 1: Add 10 µl of 1X TE.
    (or)
    Option 2: Add 10 µl of water.

    Resuspend pellet by vortexing/by shaking vigorously.
    Ensure to dry the pelletted DNA completely before attempting to resuspend.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 27 mins

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