Kafatos:Minipreps protocol

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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>5 ml 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>ddH2O</li><li>single bacterial colony</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Electrophoretic unit</li><li>Flasks of appropriate volumes</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><li>DAY 1 Inoculate 5 ml sterile LB medium (+ antibiotic) with single bacterial colony and incubate with shaking for 12 hrs(overnight) at 37°C. </li><li>DAY 2 <ol type="a"><li>Measure out 1.5 ml of culture into Eppendorf tube (1). Centrifuge at a speed of 8000 rpm for 2 mins at room temperature, gently aspirate out the supernatant and discard it. </li><li>Add 100 µl of P1 Buffer. Resuspend P1 Buffer by vortexing/by shaking vigorously. Store at room temperature for 5 mins. </li><li>Add 200 µl of P2 Buffer. Store at room temperature for at most 5 mins. </li><li>Add 150 µl of ice-cold P3 Buffer. Vortex the mixture for a few secs. Store the tube on ice for 5 - 10 mins. </li><li>Centrifuge at maximum speed for 10 mins at 4°C and aspirate out the top layer. Transfer top aqueous layer into Eppendorf tube (2). Discard bottom layer. </li><li>Add 2 volumes absolute EtOH to Eppendorf tube (2). Store the tube on ice for 10 - 20 mins. </li><li>Centrifuge at maximum speed for 10 mins at 4°C, gently aspirate out the supernatant and discard it. </li><li>Add 1 ml of 70% ethanol. Mix solution by pipetting up and down several times. </li><li>Centrifuge at maximum speed for 5 mins at 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. Do not overdo drying, or the pellet will not properly dissolve again. </li><li>Option 1: Add 30 µl of TE. (or) Option 2: Add 30 µl of ddH2O. Resuspend ddH2O 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></html>

Kafatos:Minipreps protocol

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