Small Scale Plasmid Isolation (Miniprep) protocol

Solutions/reagents: overnight cultureice-cold Solution ISolution IISolution IIIphenolchloroformRNase to a final concentration of 3µg/mlice-cold isopropanol70% ethanol</li>sterile distilled water</li></ul> Equipment: Centrifuge</li>Incubator</li>Sterile 1.5-ml microcentrifuge tubes</li></ul> Steps: <ol>  Cell Lysis  Measure out <font color=#357EC7>1.5 ml  of <font color=#357EC7>overnight culture into sterile 1.5-ml microcentrifuge tube (1). Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>3 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. </li> Add <font color=#357EC7>1.5 ml  of <font color=#357EC7>overnight culture. Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>3 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. </li> Add <font color=#357EC7>1 ml  of <font color=#357EC7>ice-cold Solution I. Resuspend pellet by vortexing/by shaking vigorously. </li> Add <font color=#357EC7>200 µl  of <font color=#357EC7>Solution II. Close the tube tightly and gently mix the contents by inverting the tube. Store the tube <font color=#357EC7>on ice  for <font color=#357EC7>5 mins . </li> Add <font color=#357EC7>150 µl  of <font color=#357EC7>Solution III. Vortex the mixture for a few secs. Store the tube <font color=#357EC7>on ice  for <font color=#357EC7>10 mins . </li> Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> and aspirate out the top layer. Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (2). Discard bottom layer. </li> </ol></li>  Phenol/Chloroform Cleanup  Add  <font color=#357EC7>0.5  volume <font color=#357EC7>phenol to sterile 1.5-ml microcentrifuge tube (2). Add <font color=#357EC7>0.5  volume <font color=#357EC7>chloroform. </li> Vortex the mixture for a few secs. Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> and aspirate out the top layer. Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (3). Discard bottom layer. </li> </ol></li>  RNase (Optional)  Measure out RNase to a final concentration of 3µg/ml into sterile 1.5-ml microcentrifuge tube (3). </li> <li>Incubate at <font color=#357EC7>37°C  for <font color=#357EC7>30 - 45 mins . <font color = "#800517">Use a water bath. </li> <li>Add  <font color=#357EC7>0.5  volume <font color=#357EC7>phenol. Add <font color=#357EC7>0.5  volume <font color=#357EC7>chloroform. Vortex the mixture for a few secs. Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> and aspirate out the top layer. Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (4). Discard bottom layer. </li> </ol></li> <li> Alcohol Precipitation / Purification  <li>Add  <font color=#357EC7>1  volume <font color=#357EC7>ice-cold isopropanol to sterile 1.5-ml microcentrifuge tube (4). </li> <li>Close the tube tightly and gently mix the contents by inverting the tube. Store the tube <font color=#357EC7>on ice  for <font color=#357EC7>10 mins . </li> <li>Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>15 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. </li> <li>Add <font color=#357EC7>200 µl  of <font color=#357EC7>70% ethanol. Mix solution by pipetting up and down several times. </li> <li>Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. </li> <li>Dry the pellet in air for at most <font color=#357EC7>30 mins . </li> <li>Add <font color=#357EC7>30 - 50 µl  of <font color=#357EC7>sterile distilled water. Resuspend pellet by vortexing/by shaking vigorously. </li> <li>Store at <font color=#357EC7>-20°C . </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 2 hrs, 21 mins