Kafatos:Minipreps protocol

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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>sterile LB medium (+ antibiotic)</li><li>P1 Buffer</li><li>P2 Buffer</li><li>ice-cold P3 Buffer</li><li>absolute EtOH</li><li>70% ethanol</li><li>TE</li><li>ddH<sub>2</sub>O</li><li>single bacterial colony</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Electrophoretic unit</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>DAY 1</font></b><br>Inoculate <font color=#357EC7>5 ml sterile LB medium (+ antibiotic)</font> with <font color=#357EC7>single bacterial colony</font> and incubate with shaking for <b><font color=#357EC7>12 hrs</font></b>(overnight) at <b><font color=#357EC7>37°C</font></b>.<br></li></p><p><li><b><font size=3>DAY 2</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>culture</font> into Eppendorf tube (1).<br>Centrifuge at a speed of <font color=#357EC7>8000 rpm</font> for <b><font color=#357EC7>2 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>P1 Buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>P2 Buffer</font>.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for at most <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>ice-cold P3 Buffer</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>5 - 10 mins</font></b>.<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> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (2).<br>Discard bottom layer.<br></li></p><p><li>Add <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>absolute EtOH</font> to Eppendorf tube (2).<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 - 20 mins</font></b>.<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>1 ml</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></ol></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 speedvac. <br></p><p><font color = "#800517"><i>Do not overdo drying, or the pellet will not properly dissolve again.</i></font><br></li></p><p><li><p><b>Option 1: </b>Add <b><font color=#357EC7>30 µl</font></b> of <font color=#357EC7>TE</font>.<br>(or)<br><b>Option 2: </b>Add <b><font color=#357EC7>30 µl</font></b> of <font color=#357EC7>ddH<sub>2</sub>O</font>.<br></p><p>Resuspend pellet by vortexing/by shaking vigorously.<br>Perform agarose gel electrophoresis of appropriate quantity of ddH<sub>2</sub>O mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 13 hrs, 7 mins</font></b></p></html>