Chromosomal DNA isolation from E. coli protocol

Solutions/reagents: culture grown in your favorite mediumice-cold Killing BufferTE10% SDS0.5M EDTAisopropanol RNase A  (25mg/ml)  <a name="proteinase K">proteinase K  (25mg/ml) </a></li>ethanol</li>3M Na-Acetate</li>distilled water</li></ul> Equipment: Centrifuge</li>Incubator</li></ul> Steps: <ol> Measure out <font color=#357EC7>1  volume <font color=#357EC7>ice-cold Killing Buffer into culture grown in your favorite medium. Vortex the mixture for a few secs. Store the tube <font color=#357EC7>on ice . <font color = "#800517">Samples should be processed as fast as possible. </li> Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>3 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. </li> Measure out <font color=#357EC7>300 µl  of <font color=#357EC7>TE into Eppendorf tube (1). Add <font color=#357EC7>40 µl  of <font color=#357EC7>10% SDS. Add <font color=#357EC7>3 µl  of <font color=#357EC7>0.5M EDTA. Resuspend pellet by vortexing/by shaking vigorously. </li> Incubate at <font color=#357EC7>65°C  for <font color=#357EC7>5 mins . </li> Add <font color=#357EC7>750 µl  of <font color=#357EC7>isopropanol. Vortex the mixture for a few secs. </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> Add <font color=#357EC7>500 µl  of <font color=#357EC7>TE. Add <font color=#357EC7>2 µl  of <a href="#RNase A" ><font color=#357EC7>RNase A </a>. Resuspend pellet by vortexing/by shaking vigorously. </li> Incubate at <font color=#357EC7>65°C  for <font color=#357EC7>30 mins . </li> Add <font color=#357EC7>2 µl  of <a href="#proteinase K" ><font color=#357EC7>proteinase K </a>. Incubate at <font color=#357EC7>37°C  for <font color=#357EC7>15 mins . </li>  phenol extract (2x phenol & 2x chlorophorm). </li>  <li>Add <font color=#357EC7>1 ml  of <font color=#357EC7>ethanol. Add <font color=#357EC7>40 µl  of <font color=#357EC7>3M Na-Acetate. Store at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> for <font color=#357EC7>12 hrs (overnight). </li> <li>Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>15 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. Add <font color=#357EC7>50 µl  of <font color=#357EC7>distilled water. Resuspend pellet by vortexing/by shaking vigorously. </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 13 hrs, 13 mins