Miniprep - Kit-free high throughput protocol: Difference between revisions
(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;">(...) |
No edit summary |
||
| Line 1: | Line 1: | ||
<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 | <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> | ||
Latest revision as of 23:50, 19 November 2009
<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>
