Miniprep - Kit-free high throughput protocol

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(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture containing your plasmid</li><li> <a name="STET buffer">STET buffer <i><br><tab><div style="margin-right: 600px;">(...)
Current revision (01:50, 20 November 2009) (view source)
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture containing your plasmid</li><li> <a name="STET buffer">STET buffer <i><br><tab><div style="margin-right: 600px;">(8% sucrose, 50 mM Tris-HCl (pH 8), 0.5% Triton X-100, 50 mM EDTA)</div></i></a></li><li>lysozyme (10mg/ml)</li><li>ice-cold isopropanol</li><li>ice-cold 80% ethanol</li><li> <a name="TE buffer">TE buffer <i><br><tab><div style="margin-right: 600px;">(10 mM Tris-HCl (pH 8), 1 mM EDTA )</div></i></a></li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Flasks of appropriate volumes</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>overnight culture containing your plasmid</font> into an Eppendorf tube.<br>Centrifuge at a speed of <font color=#357EC7>5000 rpm</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></li></p><p><li>Add <b><font color=#357EC7>300 µl</font></b> of <a href="#STET buffer" ><font color=#357EC7>STET buffer</font></a>.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>lysozyme (10mg/ml)</font>.<br>Vortex the mixture for a few secs.<br>Immerse in boiling water for <b><font color=#357EC7>40 secs</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>30 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><font color = "#800517"><i>Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick. Try to insert and remove the toothpick from the center of the tube so you don't get any cellular debris on the sides of the tube.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>300 µl</font></b> of <font color=#357EC7>ice-cold isopropanol</font>.<br><font color = "#800517"><i>This is done to precipitate the DNA.</i></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>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>ice-cold 80% ethanol</font>.<br><font color = "#800517"><i>This is to wash the pellet.</i></font><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><p>Option 1: Dry the pellet in air. <br>(or)<br>Option 2: Dry the pellet in a speedvac. <br></p><p></li></p><p><li>Add <b><font color=#357EC7>50 µl</font></b> of <a href="#TE buffer" ><font color=#357EC7>TE buffer</font></a>.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p></ol></html>
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture containing your plasmid</li><li> <a name="STET buffer">STET buffer <i><br><tab><div style="margin-right: 600px;">(8% sucrose, 50 mM Tris-HCl (pH 8), 0.5% Triton X-100, 50 mM EDTA)</div></i></a></li><li>lysozyme (10mg/ml)</li><li>ice-cold isopropanol</li><li>ice-cold 80% ethanol</li><li> <a name="TE buffer">TE buffer <i><br><tab><div style="margin-right: 600px;">(10 mM Tris-HCl (pH 8), 1 mM EDTA )</div></i></a></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>1.5 ml</font></b> of <font color=#357EC7>overnight culture containing your plasmid</font> into Eppendorf tube (1).<br>Centrifuge at a speed of <font color=#357EC7>5000 rpm</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></li></p><p><li>Measure out <b><font color=#357EC7>300 µl</font></b> of <a href="#STET buffer" ><font color=#357EC7>STET buffer</font></a> into Eppendorf tube (1).<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>lysozyme (10mg/ml)</font>.<br>Vortex the mixture for a few secs.<br>Store at <b><font color=#357EC7>100°C</font></b> for <b><font color=#357EC7>40 secs</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>30 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><font color = "#800517"><i>Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick. Try to insert and remove the toothpick from the center of the tube so you don't get any cellular debris on the sides of the tube.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>300 µl</font></b> of <font color=#357EC7>ice-cold isopropanol</font>.<br><font color = "#800517"><i>This is done to precipitate the DNA.</i></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>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>ice-cold 80% ethanol</font>.<br><font color = "#800517"><i>This is to wash the pellet.</i></font><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><p><b>Option 1: </b>Dry the pellet in air. <br>(or)<br><b>Option 2: </b>Dry the pellet in a speedvac. <br></p><p></li></p><p><li>Add <b><font color=#357EC7>50 µl</font></b> of <a href="#TE buffer" ><font color=#357EC7>TE buffer</font></a>.<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>~ 50 mins</font></b></p></html>

Current revision

Solutions/reagents:

Equipment:

  • Centrifuge
  • Eppendorf tubes

Steps:

  1. Measure out 1.5 ml of overnight culture containing your plasmid into Eppendorf tube (1).
    Centrifuge at a speed of 5000 rpm for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
  2. Measure out 300 µl of STET buffer into Eppendorf tube (1).
    Resuspend pellet by vortexing/by shaking vigorously.
  3. Add 10 µl of lysozyme (10mg/ml).
    Vortex the mixture for a few secs.
    Store at 100°C for 40 secs.
  4. Centrifuge at maximum speed for 30 mins at 4°C, gently aspirate out the supernatant and discard it.
  5. Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick. Try to insert and remove the toothpick from the center of the tube so you don't get any cellular debris on the sides of the tube.
  6. Add 300 µl of ice-cold isopropanol.
    This is done to precipitate the DNA.
  7. Centrifuge at maximum speed for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
  8. Add 200 µl of ice-cold 80% ethanol.
    This is to wash the pellet.
    Centrifuge at maximum speed for 5 mins at 4°C, gently aspirate out the supernatant and discard it.
  9. Option 1: Dry the pellet in air.
    (or)
    Option 2: Dry the pellet in a speedvac.

  10. Add 50 µl of TE buffer.
    Resuspend pellet by vortexing/by shaking vigorously.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 50 mins

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