One step 'miniprep' method for the isolation of plasmid DNA protocol

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(New page: <html> <h2>Solutions/reagents:</h2><ul type="circle"><li>overnight E.coli culture</li><li> <a name="phenol : chloroform : isoamyl alcohol(25:24:1)">phenol : chloroform : isoamyl alcohol(25...)
Current revision (02:14, 20 November 2009) (view source)
 
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<h2>Solutions/reagents:</h2><ul type="circle"><li>overnight E.coli culture</li><li> <a name="phenol : chloroform : isoamyl alcohol(25:24:1)">phenol : chloroform : isoamyl alcohol(25:24:1) <i><br><tab><div style="margin-right: 600px;">(phenol saturated with TE(10mM Tris, 7.5, 1mM EDTA) prior to mixing with chloroform and isoamylalcohol)</div></i></a></li><li>isopropanol</li><li>70% ethanol</li><li>100 µl/ml RNAse</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Flasks of appropriate volumes</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>0.5 ml</font></b> of <font color=#357EC7>overnight E.coli culture</font> into a sterile 1.5-ml microcentrifuge tube.<br><font color = "#800517"><i>We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>0.5 ml</font></b> of <a href="#phenol : chloroform : isoamyl alcohol(25:24:1)" ><font color=#357EC7>phenol : chloroform : isoamyl alcohol(25:24:1)</font></a>.<br></li></p><p><li>Vortex the mixture for <b><font color=#357EC7>1 min</font></b> .<br><font color = "#800517"><i>Vortex at maximum speed.</i></font><br><font color = "#800517"><i>Alternatively, vortex for 10 seconds and then transfer to eppendorf mixer or an over-the-top rotator for 5 minutes.</i></font><br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>12000 Xg</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>.<br></li></p><p><li><b>Meanwhile:</b><br>Set aside a fresh a sterile 1.5-ml microcentrifuge tube. Call it Tube I. <br>Measure out <b><font color=#357EC7>0.5 ml</font></b> of <font color=#357EC7>isopropanol</font> into Tube I.<br></li></p><p><li>Measure out <b><font color=#357EC7>0.45 ml</font></b> of <font color=#357EC7>top aqueous phase obtained after centrifugation</font> into Tube I.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>12000 Xg</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br><font color = "#800517"><i>Addition of salt and cooling is unnecessary.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>0.5 ml</font></b> of <font color=#357EC7>70% ethanol</font>.<br><font color = "#800517"><i>Add ethanol carefully to the side of the tube.</i></font><br>Discard solution.<br></li></p><p><li>Repeat Step 7. <br>Add <font color=#357EC7>100 µl/ml RNAse</font> to solution.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.</i></font><br></li></p></ol>
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<h2>Solutions/reagents:</h2><ul type="circle"><li>overnight E.coli culture</li><li> <a name="phenol : chloroform : isoamyl alcohol(25:24:1)">phenol : chloroform : isoamyl alcohol(25:24:1) <i><br><tab><div style="margin-right: 600px;">(phenol saturated with TE(10mM Tris, 7.5, 1mM EDTA) prior to mixing with chloroform and isoamylalcohol)</div></i></a></li><li>isopropanol</li><li>70% ethanol</li><li>100 µl/ml RNAse</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>0.5 ml</font></b> of <font color=#357EC7>overnight E.coli culture</font> into sterile 1.5-ml microcentrifuge tube (1).<br><font color = "#800517"><i>We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany.</i></font><br></li></p><p><li>Measure out <b><font color=#357EC7>0.5 ml</font></b> of <a href="#phenol : chloroform : isoamyl alcohol(25:24:1)" ><font color=#357EC7>phenol : chloroform : isoamyl alcohol(25:24:1)</font></a> into sterile 1.5-ml microcentrifuge tube (1).<br></li></p><p><li>Vortex the mixture for <b><font color=#357EC7>1 min</font></b> .<br><font color = "#800517"><i>Vortex at maximum speed.</i></font><br><font color = "#800517"><i>Alternatively, vortex for 10 seconds and then transfer to eppendorf mixer or an over-the-top rotator for 5 minutes.</i></font><br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>12000 Xg</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>.<br></li></p><p><li><b>Meanwhile:</b><br>Set aside a fresh sterile 1.5-ml microcentrifuge tube (2). Call it Tube I. <br>Measure out <b><font color=#357EC7>0.5 ml</font></b> of <font color=#357EC7>isopropanol</font> into Tube I.<br></li></p><p><li>Add <b><font color=#357EC7>0.45 ml</font></b> of <font color=#357EC7>top aqueous phase obtained after centrifugation</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>12000 Xg</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br><font color = "#800517"><i>Addition of salt and cooling is unnecessary.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>0.5 ml</font></b> of <font color=#357EC7>70% ethanol</font>.<br><font color = "#800517"><i>Add ethanol carefully to the side of the tube.</i></font><br>Discard solution.<br></li></p><p><li>Repeat Step 7. <br>Add <font color=#357EC7>100 µl/ml RNAse</font> to solution.<br>Resuspend pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.</i></font><br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 11 mins</font></b></p>
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Current revision

Solutions/reagents:

Equipment:

  • Centrifuge
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Measure out 0.5 ml of overnight E.coli culture into sterile 1.5-ml microcentrifuge tube (1).
    We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany.
  2. Measure out 0.5 ml of phenol : chloroform : isoamyl alcohol(25:24:1) into sterile 1.5-ml microcentrifuge tube (1).
  3. Vortex the mixture for 1 min .
    Vortex at maximum speed.
    Alternatively, vortex for 10 seconds and then transfer to eppendorf mixer or an over-the-top rotator for 5 minutes.
  4. Centrifuge at a speed of 12000 Xg for 5 mins at room temperature.
  5. Meanwhile:
    Set aside a fresh sterile 1.5-ml microcentrifuge tube (2). Call it Tube I.
    Measure out 0.5 ml of isopropanol into Tube I.
  6. Add 0.45 ml of top aqueous phase obtained after centrifugation.
    Vortex the mixture for a few secs.
    Centrifuge at a speed of 12000 Xg for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
    Addition of salt and cooling is unnecessary.
  7. Add 0.5 ml of 70% ethanol.
    Add ethanol carefully to the side of the tube.
    Discard solution.
  8. Repeat Step 7.
    Add 100 µl/ml RNAse to solution.
    Resuspend pellet by vortexing/by shaking vigorously.
    About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 11 mins

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