Miniprep - GET Buffer protocol

Solutions/reagents: overnight culture  ice-cold GET buffer  (50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl pH 8) 0.2M NaOH stored at room temperature  ice-cold potassium acetate solution  (3 M potassium acetate, 1.8 M acetic acid, no pH adjustment) 95% / 100% ethanol70% EtOHTE buffer</li>distilled water</li> <a name="PCA solution">PCA solution  ((optional)50 parts phenol, 49 parts chloroform, and 1 part amyl-alcohol) </a></li>SDS</li> lysozyme  (optional) </a></li></ul> Equipment: Centrifuge</li>Sterile 2-ml microcentrifuge tubes</li></ul> Steps: <ol> Measure out <font color=#357EC7>2 ml  of <font color=#357EC7>overnight culture into sterile 2-ml microcentrifuge tube (1). </li> Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>1 min  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. </li> Measure out <font color=#357EC7>100 µl  of <a href="#ice-cold GET buffer" ><font color=#357EC7>ice-cold GET buffer </a> into sterile 2-ml microcentrifuge tube (1). Resuspend pellet by vortexing/by shaking vigorously. </li>  (Optional)  Add <font color=#357EC7> 10 mg  of <a href="#lysozyme" ><font color=#357EC7>lysozyme </a>. Store at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> for <font color=#357EC7>30 mins . <font color = "#800517">This step is essential for lysing gram-positive cells. </li> Measure out <font color=#357EC7>200 µl  of <font color=#357EC7>0.2M NaOH into sterile 2-ml microcentrifuge tube (2). Add <font color=#357EC7> 2 mg  of <font color=#357EC7>SDS. Vortex the mixture for a few secs. </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. <font color = "#800517">''DO NOT VORTEX! The solution should become clear.'' </li> Add <font color=#357EC7>150 µl  of <a href="#ice-cold potassium acetate solution" ><font color=#357EC7>ice-cold potassium acetate solution </a>. Close the tube tightly and gently mix the contents by inverting the tube. <font color = "#800517">''DO NOT VORTEX! A precipitate should form.'' </li> Store the tube <font color=#357EC7>on ice  for <font color=#357EC7>3 - 5 mins . </li> Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>10 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> and aspirate out <font color=#357EC7>400 µl  of top layer. Transfer top aqueous layer into sterile 2-ml microcentrifuge tube (3). Discard bottom layer. <font color = "#800517">DO NOT PICK UP ANY PRECIPITATE!!! </li>  (Optional)  Measure out <font color=#357EC7>400 µl  of <a href="#PCA solution" ><font color=#357EC7>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 <font color=#357EC7>maximum speed for <font color=#357EC7>3 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> and aspirate out the top layer. Transfer top aqueous layer into sterile 2-ml microcentrifuge tube (4). Discard bottom layer. <font color = "#800517">This helps remove any residual proteins. </li> Measure out <font color=#357EC7>900 µl  of <font color=#357EC7>95% / 100% ethanol into sterile 2-ml microcentrifuge tube (4). <font color = "#800517">This is to precipitate the plasmid DNA. </li> <li>Store at <font color=#357EC7>-80°C  for <font color=#357EC7>30 mins . <font color = "#800517">Use the -80°C freezer. </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. Store at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> for <font color=#357EC7>3 mins . </li> <li>Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>3 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. <font color = "#800517">Make sure the pellet is toward the outside. </li> <li>Dry the pellet in air for <font color=#357EC7>10 - 15 mins . </li> <li><font color = "#800517">Make sure the pellet is completely dry before this step.  Option 1: Add <font color=#357EC7>20 µl  of <font color=#357EC7>TE buffer. (or) Option 2: Add <font color=#357EC7>20 µl  of <font color=#357EC7>distilled water. Resuspend pellet by vortexing/by shaking vigorously. <font color = "#800517"> 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 :<font color=#357EC7>~ 1 hr, 50 mins