Miniprep - GET Buffer protocol

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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li> <a name="ice-cold GET buffer"> ice-cold GET buffer <tab><div style="margin-right: 600px;">(50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl pH 8)</div></a></li><li>0.2M NaOH stored at room temperature</li><li> <a name="ice-cold potassium acetate solution"> ice-cold potassium acetate solution <tab><div style="margin-right: 600px;">(3 M potassium acetate, 1.8 M acetic acid, no pH adjustment)</div></a></li><li>95% / 100% ethanol</li><li>70% EtOH</li><li>TE buffer</li><li>distilled water</li><li> <a name="PCA solution">PCA solution <tab><div style="margin-right: 600px;">((optional)50 parts phenol, 49 parts chloroform, and 1 part amyl-alcohol)</div></a></li><li>SDS</li><li> <a name="lysozyme">lysozyme <tab>(optional) </a></li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Sterile 2-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><li>Measure out 2 ml of overnight culture into sterile 2-ml microcentrifuge tube (1). </li><li>Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it. </li><li>Measure out 100 µl of <a href="#ice-cold GET buffer" >ice-cold GET buffer</a> into sterile 2-ml microcentrifuge tube (1). Resuspend pellet by vortexing/by shaking vigorously. </li>(Optional) Add 10 mg of <a href="#lysozyme" >lysozyme</a>. Store at room temperature for 30 mins. This step is essential for lysing gram-positive cells. </li><li>Measure out 200 µl of 0.2M NaOH into sterile 2-ml microcentrifuge tube (2). Add 2 mg of SDS. Vortex the mixture for a few secs. </li><li>Measure out alkaline SDS solution into sterile 2-ml microcentrifuge tube (1). Close the tube tightly and gently mix the contents by inverting the tube. DO NOT VORTEX! The solution should become clear. </li><li>Add 150 µl of <a href="#ice-cold potassium acetate solution" >ice-cold potassium acetate solution</a>. Close the tube tightly and gently mix the contents by inverting the tube. DO NOT VORTEX! A precipitate should form. </li><li>Store the tube on ice for 3 - 5 mins. </li><li>Centrifuge at maximum speed for 10 mins at room temperature and aspirate out 400 µl of top layer. Transfer top aqueous layer into sterile 2-ml microcentrifuge tube (3). Discard bottom layer. DO NOT PICK UP ANY PRECIPITATE!!! </li>(Optional) Measure out 400 µl of <a href="#PCA solution" >PCA solution</a> into sterile 2-ml microcentrifuge tube (3). Close the tube tightly and gently mix the contents by inverting the tube. Centrifuge at maximum speed for 3 mins at room temperature and aspirate out the top layer. Transfer top aqueous layer into sterile 2-ml microcentrifuge tube (4). Discard bottom layer. This helps remove any residual proteins. </li><li>Measure out 900 µl of 95% / 100% ethanol into sterile 2-ml microcentrifuge tube (4). This is to precipitate the plasmid DNA. </li><li>Store at -80°C for 30 mins. Use the -80°C freezer. </li><li>Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it. </li><li>Add 1 ml of 70% EtOH. Store at room temperature for 3 mins. </li><li>Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it. Make sure the pellet is toward the outside. </li><li>Dry the pellet in air for 10 - 15 mins. </li><li>Make sure the pellet is completely dry before this step. Option 1: Add 20 µl of TE buffer. (or) Option 2: Add 20 µl of distilled water. Resuspend pellet by vortexing/by shaking vigorously. The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse. </li></ol></ul></ul>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 50 mins</html>

Miniprep - GET Buffer protocol

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