Erman's Lab:DNA Miniprep with Alkaline Lysis protocol

Solutions/reagents: LB (+ antibiotics) Alkaline Lysis SLN1  (20mM Tris(pH 8), 50mM Glucose, 10mM EDTA)  freshly prepared AL2  (0.2N NaOH, 1% SDS)  AL3  (3M KAc, glacial acetic acid) isopropanol stored at room temperature70% EtOH</li>TE</li>water</li>single colony</li></ul> Equipment: Incubator</li>Centrifuge</li>Eppendorf tubes</li></ul> Steps: <ol> Inoculate <font color=#357EC7>2 ml LB (+ antibiotics) with <font color=#357EC7>single colony and incubate with shaking for <font color=#357EC7>12 hrs (overnight) at <font color=#357EC7>37°C . </li> Measure out <font color=#357EC7>2 ml  of <font color=#357EC7>culture into Eppendorf tube (1). </li> Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>1.5 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. </li> <font color = "#800517">Leave pellet as dry as possible. </li>  Add <font color=#357EC7>100 µl  of <a href="#Alkaline Lysis SLN1" ><font color=#357EC7>Alkaline Lysis SLN1 </a>. Resuspend pellet by vortexing/by shaking vigorously. </li> Add <font color=#357EC7>200 µl  of <a href="#freshly prepared AL2" ><font color=#357EC7>freshly prepared AL2 </a>. Close the tube tightly and invert the tube <font color=#357EC7>5 - 6 times . <font color = "#800517">Do not vortex! </li> Store the tube <font color=#357EC7>on ice . </li> Add <font color=#357EC7>300 µl  of <a href="#AL3" ><font color=#357EC7>AL3 </a>. Close the tube tightly and gently mix the contents by inverting the tube. </li> Incubate on <font color=#357EC7><b><font color=#357EC7>ice  </b> for <font color=#357EC7>5 mins . </li> <li>Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>5 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> <li>Measure out <font color=#357EC7>900 µl  of <font color=#357EC7>isopropanol into Eppendorf tube (2). Vortex the mixture for a few secs. </li> <li>Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>10 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. </li> <li>Add <font color=#357EC7>1 ml  of <font color=#357EC7>70% EtOH. Vortex the mixture for a few secs. </li> <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> <li>Dry the pellet in air. </li> <li> Option 1: Add <font color=#357EC7>50 µl  of <font color=#357EC7>TE. (or) Option 2: Add <font color=#357EC7>50 µl  of <font color=#357EC7>water. Dissolve the pellet in the solution. </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 12 hrs, 26 mins