Difference between revisions of "Small Scale Plasmid Isolation (Miniprep) protocol"

From OpenWetWare
Jump to: navigation, search
 
Line 1: Line 1:
 
<html>
 
<html>
<h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li>ice-cold Solution I</li><li>Solution II</li><li>Solution III</li><li>phenol</li><li>chloroform</li><li>RNase to a final concentration of 3µg/ml</li><li>ice-cold isopropanol</li><li>70% ethanol</li><li>sterile distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Cell Lysis</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>overnight culture</font> into sterile 1.5-ml microcentrifuge tube (1).<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>1.5 ml</font></b> of <font color=#357EC7>overnight culture</font>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>100 µl</font></b> of <font color=#357EC7>ice-cold Solution I</font>.<br>Resuspend ice-cold Solution I by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>Solution II</font>.<br>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>Solution III</font>.<br>Vortex the mixture for a few secs.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 mins</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></li></p></ol></li></p><p><li><b><font size=3>Phenol/Chloroform Cleanup</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font> to sterile 1.5-ml microcentrifuge tube (2).<br>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br></li></p><p><li>Vortex the mixture for a few secs.<br>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><b><font size=3>RNase (Optional)</font></b><br><ol type="a"><p><li>Measure out RNase to a final concentration of 3µg/ml into sterile 1.5-ml microcentrifuge tube (3).<br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>30 - 45 mins</font></b>.<br><font color = "#800517"><i>Use a water bath.</i></font><br></li></p><p><li>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font>.<br>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br>Vortex the mixture for a few secs.<br>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 (4).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Alcohol Precipitation / Purification</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>ice-cold isopropanol</font> to sterile 1.5-ml microcentrifuge tube (4).<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</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>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Mix solution by pipetting up and down several times.<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>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air for at most <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>30 - 50 µl</font></b> of <font color=#357EC7>sterile distilled water</font>.<br>Resuspend sterile distilled water by vortexing/by shaking vigorously.<br></li></p><p><li>Store at <b><font color=#357EC7>-20°C</font></b>.<br></li></p></ol>
+
<h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li>ice-cold Solution I</li><li>Solution II</li><li>Solution III</li><li>phenol</li><li>chloroform</li><li>RNase to a final concentration of 3µg/ml</li><li>ice-cold isopropanol</li><li>70% ethanol</li><li>sterile distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Cell Lysis</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>overnight culture</font> into sterile 1.5-ml microcentrifuge tube (1).<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>1.5 ml</font></b> of <font color=#357EC7>overnight culture</font>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>1 ml</font></b> of <font color=#357EC7>ice-cold Solution I</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>Solution II</font>.<br>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>Solution III</font>.<br>Vortex the mixture for a few secs.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 mins</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></li></p></ol></li></p><p><li><b><font size=3>Phenol/Chloroform Cleanup</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font> to sterile 1.5-ml microcentrifuge tube (2).<br>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br></li></p><p><li>Vortex the mixture for a few secs.<br>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><b><font size=3>RNase (Optional)</font></b><br><ol type="a"><p><li>Measure out RNase to a final concentration of 3µg/ml into sterile 1.5-ml microcentrifuge tube (3).<br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>30 - 45 mins</font></b>.<br><font color = "#800517"><i>Use a water bath.</i></font><br></li></p><p><li>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font>.<br>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br>Vortex the mixture for a few secs.<br>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 (4).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Alcohol Precipitation / Purification</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>ice-cold isopropanol</font> to sterile 1.5-ml microcentrifuge tube (4).<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</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>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Mix solution by pipetting up and down several times.<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>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air for at most <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>30 - 50 µl</font></b> of <font color=#357EC7>sterile distilled water</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Store at <b><font color=#357EC7>-20°C</font></b>.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 2 hrs, 21 mins</font></b></p>
 
</html>
 
</html>

Latest revision as of 23:10, 19 November 2009

<html> <h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li>ice-cold Solution I</li><li>Solution II</li><li>Solution III</li><li>phenol</li><li>chloroform</li><li>RNase to a final concentration of 3µg/ml</li><li>ice-cold isopropanol</li><li>70% ethanol</li><li>sterile distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Cell Lysis</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>overnight culture</font> into sterile 1.5-ml microcentrifuge tube (1).<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>1.5 ml</font></b> of <font color=#357EC7>overnight culture</font>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>1 ml</font></b> of <font color=#357EC7>ice-cold Solution I</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>Solution II</font>.<br>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>Solution III</font>.<br>Vortex the mixture for a few secs.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 mins</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></li></p></ol></li></p><p><li><b><font size=3>Phenol/Chloroform Cleanup</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font> to sterile 1.5-ml microcentrifuge tube (2).<br>Add <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br></li></p><p><li>Vortex the mixture for a few secs.<br>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><b><font size=3>RNase (Optional)</font></b><br><ol type="a"><p><li>Measure out RNase to a final concentration of 3µg/ml into sterile 1.5-ml microcentrifuge tube (3).<br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>30 - 45 mins</font></b>.<br><font color = "#800517"><i>Use a water bath.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font>.<br>Add <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br>Vortex the mixture for a few secs.<br>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 (4).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Alcohol Precipitation / Purification</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>ice-cold isopropanol</font> to sterile 1.5-ml microcentrifuge tube (4).<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</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>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Mix solution by pipetting up and down several times.<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>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air for at most <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>30 - 50 µl</font></b> of <font color=#357EC7>sterile distilled water</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Store at <b><font color=#357EC7>-20°C</font></b>.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 2 hrs, 21 mins</font></b></p> </html>