Kafatos:Minipreps protocol

Solutions/reagents: sterile LB medium (+ antibiotic)P1 BufferP2 Bufferice-cold P3 Bufferabsolute EtOH70% ethanolTEddH2Osingle bacterial colony</li></ul> Equipment: Incubator</li>Centrifuge</li>Electrophoretic unit</li>Eppendorf tubes</li></ul> Steps: <ol>  DAY 1  Inoculate <font color=#357EC7>5 ml sterile LB medium (+ antibiotic) with <font color=#357EC7>single bacterial colony and incubate with shaking for <font color=#357EC7>12 hrs (overnight) at <font color=#357EC7>37°C . </li>  DAY 2  Measure out <font color=#357EC7>1.5 ml  of <font color=#357EC7>culture into Eppendorf tube (1). Centrifuge at a speed of <font color=#357EC7>8000 rpm for <font color=#357EC7>2 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. </li> Add <font color=#357EC7>100 µl  of <font color=#357EC7>P1 Buffer. Resuspend pellet by vortexing/by shaking vigorously. Store at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> for <font color=#357EC7>5 mins . </li> Add <font color=#357EC7>200 µl  of <font color=#357EC7>P2 Buffer. Store at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> for at most <font color=#357EC7>5 mins . </li> Add <font color=#357EC7>150 µl  of <font color=#357EC7>ice-cold P3 Buffer. Vortex the mixture for a few secs. Store the tube <font color=#357EC7>on ice  for <font color=#357EC7>5 - 10 mins . </li> Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>10 mins  at <font color=#357EC7>4°C  and aspirate out the top layer. Transfer top aqueous layer into Eppendorf tube (2). Discard bottom layer. </li> Add  <font color=#357EC7>2  volumes <font color=#357EC7>absolute EtOH to Eppendorf tube (2). Store the tube <font color=#357EC7>on ice  for <font color=#357EC7>10 - 20 mins . </li> Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>10 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. </li> Add <font color=#357EC7>1 ml  of <font color=#357EC7>70% ethanol. Mix solution by pipetting up and down several times. </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> </ol></li> <li> Option 1: Dry the pellet in air. (or) Option 2: Dry the pellet in speedvac. <font color = "#800517">Do not overdo drying, or the pellet will not properly dissolve again. </li> <li> Option 1: Add <font color=#357EC7>30 µl  of <font color=#357EC7>TE. (or) Option 2: Add <font color=#357EC7>30 µl  of <font color=#357EC7>ddH2O. Resuspend pellet by vortexing/by shaking vigorously. Perform agarose gel electrophoresis of appropriate quantity of ddH2O mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product. </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 13 hrs, 7 mins