Small Scale Plasmid Isolation (Miniprep) protocol

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<html> <h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li>ice-cold Solution I</li><li>Solution II</li><li>Solution III</li><li>phenol</li><li>chloroform</li><li>RNase to a final concentration of 3µg/ml</li><li>ice-cold isopropanol</li><li>70% ethanol</li><li>sterile distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><li>Cell Lysis <ol type="a"><li>Measure out 1.5 ml of overnight culture into sterile 1.5-ml microcentrifuge tube (1). Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it. </li><li>Add 1.5 ml of overnight culture. Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it. </li><li>Add 100 µl of ice-cold Solution I. Resuspend ice-cold Solution I by vortexing/by shaking vigorously. </li><li>Add 200 µl of Solution II. Close the tube tightly and gently mix the contents by inverting the tube. Store the tube on ice for 5 mins. </li><li>Add 150 µl of Solution III. Vortex the mixture for a few secs. Store the tube on ice for 10 mins. </li><li>Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer. Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (2). Discard bottom layer. </li></ol></li><li>Phenol/Chloroform Cleanup <ol type="a"><li>Add 0.5 volume phenol to sterile 1.5-ml microcentrifuge tube (2). Add 0.5 volume chloroform. </li><li>Vortex the mixture for a few secs. Centrifuge at maximum speed for 5 mins at room temperature 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) <ol type="a"><li>Measure out RNase to a final concentration of 3µg/ml into sterile 1.5-ml microcentrifuge tube (3). </li><li>Incubate at 37°C for 30 - 45 mins. Use a water bath. </li><li>Add 0.5 volume phenol. Add 0.5 volume chloroform. Vortex the mixture for a few secs. Centrifuge at maximum speed for 5 mins at room temperature 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 <ol type="a"><li>Add 1 volume 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 on ice for 10 mins. </li><li>Centrifuge at maximum speed for 15 mins at room temperature, gently aspirate out the supernatant and discard it. </li><li>Add 200 µl 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><li>Dry the pellet in air for at most 30 mins. </li><li>Add 30 - 50 µl of sterile distilled water. Resuspend sterile distilled water by vortexing/by shaking vigorously. </li><li>Store at -20°C. </li></ol> </html>

Small Scale Plasmid Isolation (Miniprep) protocol

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