Size selective DNA precipitation protocol

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(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="DNA sample">DNA sample <i><br><tab><div style="margin-right: 600px;">(DNA to be separated (e.g. PCR reaction mixture))</di...)
Current revision (02:53, 20 November 2009) (view source)
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="DNA sample">DNA sample <i><br><tab><div style="margin-right: 600px;">(DNA to be separated (e.g. PCR reaction mixture))</div></i></a></li><li> <a name="PEG/MgCl2">PEG/MgCl2 <i><br><tab><div style="margin-right: 600px;">(30% (w/v) PEG 8000/30 mM MgCl2 (concentration of PEG 8000 can be varied to shift the size of the percipitated DNA. The concentration used here will remove DNA fragments with less than 300bp))</div></i></a></li><li> <a name="TE buffer">TE buffer <i><br><tab><div style="margin-right: 600px;">(10 mM TRIS-HCl, 1 mM EDTA, pH 8.0)</div></i></a></li><li>buffer of choice</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>50 µl</font></b> of <a href="#DNA sample" ><font color=#357EC7>DNA sample</font></a> into Eppendorf tube (1).<br>Add <b><font color=#357EC7>150 µl</font></b> of <a href="#TE buffer" ><font color=#357EC7>TE buffer</font></a>.<br>Gently tap the mixture for a few secs.<br></li></p><p><li>Add <b><font color=#357EC7>10 µl</font></b> of <a href="#PEG/MgCl2" ><font color=#357EC7>PEG/MgCl2</font></a>.<br></li></p><p><li>Vortex the mixture for a few secs.<br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>10000 Xg</font> for <b><font color=#357EC7>15 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><font color = "#800517"><i>Carefully remove supernatant not to disturb the pellet, which will be invisible.</i></font><br></li></p><p><li>Add <font color=#357EC7>buffer of choice</font> to pellet.<br><font color = "#800517"><i>Add appropriate volume of buffer.</i></font><br>Dissolve the pellet in the solution.<br></li></p></ol></html>
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="DNA sample">DNA sample <i><br><tab><div style="margin-right: 600px;">(DNA to be separated (e.g. PCR reaction mixture))</div></i></a></li><li> <a name="PEG/MgCl2">PEG/MgCl2 <i><br><tab><div style="margin-right: 600px;">(30% (w/v) PEG 8000/30 mM MgCl2 (concentration of PEG 8000 can be varied to shift the size of the percipitated DNA. The concentration used here will remove DNA fragments with less than 300bp))</div></i></a></li><li> <a name="TE buffer">TE buffer <i><br><tab><div style="margin-right: 600px;">(10 mM TRIS-HCl, 1 mM EDTA, pH 8.0)</div></i></a></li><li>buffer of choice</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>50 µl</font></b> of <a href="#DNA sample" ><font color=#357EC7>DNA sample</font></a> into Eppendorf tube (1).<br>Measure out <b><font color=#357EC7>150 µl</font></b> of <a href="#TE buffer" ><font color=#357EC7>TE buffer</font></a> into DNA sample.<br>Gently tap the mixture for a few secs.<br></li></p><p><li>Measure out <b><font color=#357EC7>10 µl</font></b> of <a href="#PEG/MgCl2" ><font color=#357EC7>PEG/MgCl2</font></a> into Eppendorf tube (1).<br></li></p><p><li>Vortex the mixture for a few secs.<br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>10000 Xg</font> for <b><font color=#357EC7>15 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><font color = "#800517"><i>Carefully remove supernatant not to disturb the pellet, which will be invisible.</i></font><br></li></p><p><li>Add <font color=#357EC7>buffer of choice</font> to pellet.<br><font color = "#800517"><i>Add appropriate volume of buffer.</i></font><br>Dissolve the pellet in the solution.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 15 mins</font></b></p></html>

Current revision

Solutions/reagents:

Equipment:

  • Centrifuge
  • Eppendorf tubes

Steps:

  1. Measure out 50 µl of DNA sample into Eppendorf tube (1).
    Measure out 150 µl of TE buffer into DNA sample.
    Gently tap the mixture for a few secs.
  2. Measure out 10 µl of PEG/MgCl2 into Eppendorf tube (1).
  3. Vortex the mixture for a few secs.
  4. Centrifuge at a speed of 10000 Xg for 15 mins at room temperature, gently aspirate out the supernatant and discard it.
  5. Carefully remove supernatant not to disturb the pellet, which will be invisible.
  6. Add buffer of choice to pellet.
    Add appropriate volume of buffer.
    Dissolve the pellet in the solution.

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

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