DNA extraction from tissue protocol

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(New page: <html> <h2>Solutions/reagents:</h2><ul type="circle"><li>ReagentB</li><li>ProteinaseK</li><li> <a name="DNA extraction buffer">DNA extraction buffer <i><br><tab><div style="margin-right: 6...)
Current revision (02:42, 20 November 2009) (view source)
 
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<h2>Solutions/reagents:</h2><ul type="circle"><li>ReagentB</li><li>ProteinaseK</li><li> <a name="DNA extraction buffer">DNA extraction buffer <i><br><tab><div style="margin-right: 600px;">(98µl ReagentB + 2µl ProteinaseK, mix fresh)</div></i></a></li><li>24:1 Chloroform:Isoamyl alcohol</li><li> <a name="PCI mix">PCI mix <i><br><tab><div style="margin-right: 600px;">(One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol, shake thoroughly to make emulsion)</div></i></a></li><li>100% EtOH</li><li>3M sodium acetate pH 5.0</li><li>70% EtOH</li><li>MilliQ water</li><li>small piece of tissue or embryo</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>Proteinase K digestion</font></b><br><ol type="a"><p><li>Measure out <a href="#DNA extraction buffer" ><font color=#357EC7>DNA extraction buffer</font></a> into a sterile 1.5-ml microcentrifuge tube.<br><font color = "#800517"><i>100 µl is enough for a small pea size chunk of tissue or one embryo.</i></font><br></li></p><p><li>Add <font color=#357EC7>small piece of tissue or embryo</font> to DNA extraction buffer.<br></li></p><p><li>Incubate at <b><font color=#357EC7>50°C</font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></li></p></ol></li></p><p><li><b><font size=3>Phenol/chloroform/isoamyl (PCI)</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>1</font></b> volume <a href="#PCI mix" ><font color=#357EC7>PCI mix</font></a>.<br></li></p><p><li>Vortex the mixture for <b><font color=#357EC7>10 secs</font></b> .<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></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 = "red"><i>Repeat this step as needed.</i></font><br></li></p><p><li>Add  <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>24:1 Chloroform:Isoamyl alcohol</font>.<br></li></p><p><li>Vortex the mixture for <b><font color=#357EC7>10 secs</font></b> .<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></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>Ethanol precipitation</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>100% EtOH</font>.<br></li></p><p><li>Add  <b><font color=#357EC7>0.1</font></b> volume <font color=#357EC7>3M sodium acetate pH 5.0</font>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>70% EtOH</font>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air. <br></li></p><p><li>Add <font color=#357EC7>MilliQ water</font> to pellet.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p></ol>
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<h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="DNA extraction buffer">DNA extraction buffer <i><br><tab><div style="margin-right: 600px;">(98µl ReagentB + 2µl ProteinaseK, mix fresh)</div></i></a></li><li>24:1 Chloroform:Isoamyl alcohol</li><li> <a name="PCI mix">PCI mix <i><br><tab><div style="margin-right: 600px;">(One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol, shake thoroughly to make emulsion)</div></i></a></li><li>100% EtOH</li><li>3M sodium acetate pH 5.0</li><li>70% EtOH</li><li>MilliQ water</li><li>small piece of tissue or embryo</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>Proteinase K digestion</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>100 µl</font></b> of <a href="#DNA extraction buffer" ><font color=#357EC7>DNA extraction buffer</font></a> into sterile 1.5-ml microcentrifuge tube (1).<br><font color = "#800517"><i>100 µl is enough for a small pea size chunk of tissue or one embryo.</i></font><br></li></p><p><li>Add <font color=#357EC7>small piece of tissue or embryo</font>.<br></li></p><p><li>Incubate at <b><font color=#357EC7>50°C</font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></li></p></ol></li></p><p><li><b><font size=3>Phenol/chloroform/isoamyl (PCI)</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>1</font></b> volume <a href="#PCI mix" ><font color=#357EC7>PCI mix</font></a>.<br></li></p><p><li>Vortex the mixture for <b><font color=#357EC7>10 secs</font></b> .<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (2).<br>Discard bottom layer.<br><font color = "red"><i>Repeat this step as needed.</i></font><br></li></p><p><li>Add  <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>24:1 Chloroform:Isoamyl alcohol</font> to sterile 1.5-ml microcentrifuge tube (2).<br></li></p><p><li>Vortex the mixture for <b><font color=#357EC7>10 secs</font></b> .<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></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>Ethanol precipitation</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>100% EtOH</font> to sterile 1.5-ml microcentrifuge tube (3).<br></li></p><p><li>Add  <b><font color=#357EC7>0.1</font></b> volume <font color=#357EC7>3M sodium acetate pH 5.0</font>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>70% EtOH</font>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air. <br></li></p><p><li>Add <font color=#357EC7>MilliQ water</font> to pellet.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 12 hrs, 22 mins</font></b></p>
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Current revision

Solutions/reagents:

Equipment:

  • Incubator
  • Centrifuge
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Proteinase K digestion

    1. Measure out 100 µl of DNA extraction buffer into sterile 1.5-ml microcentrifuge tube (1).
      100 µl is enough for a small pea size chunk of tissue or one embryo.
    2. Add small piece of tissue or embryo.
    3. Incubate at 50°C for 12 hrs(overnight).
  2. Phenol/chloroform/isoamyl (PCI)

    1. Add 1 volume PCI mix.
    2. Vortex the mixture for 10 secs .
    3. Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (2).
      Discard bottom layer.
      Repeat this step as needed.
    4. Add 1 volume 24:1 Chloroform:Isoamyl alcohol to sterile 1.5-ml microcentrifuge tube (2).
    5. Vortex the mixture for 10 secs .
    6. Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (3).
      Discard bottom layer.
  3. Ethanol precipitation

    1. Add 2 volumes 100% EtOH to sterile 1.5-ml microcentrifuge tube (3).
    2. Add 0.1 volume 3M sodium acetate pH 5.0.
    3. Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
    4. Add 150 µl of 70% EtOH.
    5. Centrifuge at maximum speed for 2 mins at room temperature, gently aspirate out the supernatant and discard it.
    6. Dry the pellet in air.
    7. Add MilliQ water to pellet.
      Resuspend pellet by vortexing/by shaking vigorously.

    TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 12 hrs, 22 mins

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