Erman's Lab:DNA Miniprep with Alkaline Lysis protocol

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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>2 ml of LB (+ antibiotics)</li><li> <a name="Alkaline Lysis SLN1">Alkaline Lysis SLN1 <tab><div style="margin-right: 600px;">(20mM Tris(pH 8), 50mM Glucose, 10mM EDTA)</div></a></li><li> <a name="freshly prepared AL2">freshly prepared AL2 <tab><div style="margin-right: 600px;">(0.2N NaOH, 1% SDS)</div></a></li><li> <a name="AL3">AL3 <tab><div style="margin-right: 600px;">(3M KAc, glacial acetic acid)</div></a></li><li>isopropanol stored at room temperature</li><li>70% EtOH</li><li>TE</li><li>water</li><li>single colony</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Flasks of appropriate volumes</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><li>Inoculate 2 ml of LB (+ antibiotics) with single colony and incubate with shaking for 12 hrs(overnight) at 37°C. </li><li>Measure out 2 ml of culture into Eppendorf tube (1). </li><li>Centrifuge at maximum speed for 1.5 mins at 4°C, gently aspirate out the supernatant and discard it. </li><li>Leave pellet as dry as possible. </li><li>Add 100 µl of <a href="#Alkaline Lysis SLN1" >Alkaline Lysis SLN1</a>. Resuspend Alkaline Lysis SLN1 by vortexing/by shaking vigorously. </li><li>Add 200 µl of <a href="#freshly prepared AL2" >freshly prepared AL2</a>. Close the tube tightly and invert the tube 5 - 6 times. Do not vortex! </li><li>Store the tube on ice. </li><li>Add 300 µl of <a href="#AL3" >AL3</a>. Close the tube tightly and gently mix the contents by inverting the tube. </li><li>Incubate on ice for 5 mins. </li><li>Centrifuge at maximum speed for 5 mins at 4°C and aspirate out the top layer. Transfer top aqueous layer into Eppendorf tube (2). Discard bottom layer. </li><li>Measure out 900 µl of isopropanol into Eppendorf tube (2). Vortex the mixture for a few secs. </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. Vortex the mixture for a few secs. </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. </li><li>Option 1: Add 50 µl of TE. (or)Option 2: Add 50 µl of water. Dissolve the pellet in the solution. </li></ol></html>

Erman's Lab:DNA Miniprep with Alkaline Lysis protocol

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