Maxiprep of plasmid DNA from E.coli protocol

Solutions/reagents: LB broth + selective marker50% sterile glycerol TEG  (25mM Tris-Cl, 10mM EDTA, 50mM dextrose) 20 mg/ml lysozyme10% SDS4M NaOHautoclaved water <a name="Solution 3">Solution 3  (3M potassium-acetate, 2M acetic acid -- glacial is 17M) </a></li>isopropanol</li>70% ethanol</li>TE buffer</li>5M LiCl</li>1 mg/ml RNaseA</li> <a name="phenol: chloroform: isoamyl alcohol">phenol: chloroform: isoamyl alcohol  (25:24:1) </a></li> <a name="chloroform: isoamyl alcohol">chloroform: isoamyl alcohol  (24:1) </a></li>straight ethanol</li>3M sodium acetate</li>a single colony of E. coli</li></ul> Equipment: Incubator</li>Centrifuge</li>Eppendorf tubes</li>Oakridge tubes</li></ul> Steps: <ol> Inoculate <font color=#357EC7>50 ml LB broth + selective marker with <font color=#357EC7>a single colony of E. coli and incubate with shaking for <font color=#357EC7>12 hrs (overnight) at <font color=#357EC7>37°C . </li> <li>Measure out <font color=#357EC7>850 µl  of <font color=#357EC7>culture into Eppendorf tube (1). Add <font color=#357EC7>150 µl  of <font color=#357EC7>50% sterile glycerol. Store at <font color=#357EC7>-80°C . Measure out <font color=#357EC7>850 µl  of <font color=#357EC7>culture into Eppendorf tube (2). Add <font color=#357EC7>150 µl  of <font color=#357EC7>50% sterile glycerol. Store at <font color=#357EC7>-80°C . </li> <li>Measure out as much culture as will fit into Oakridge tube (1). Centrifuge at a speed of <font color=#357EC7>5800 Xg for <font color=#357EC7>10 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. Add <font color=#357EC7>rest of the culture to pellet. Centrifuge at a speed of <font color=#357EC7>5800 Xg for <font color=#357EC7>10 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. Add <font color=#357EC7>1 ml  of <a href="#TEG" ><font color=#357EC7>TEG </a>. Resuspend pellet by vortexing/by shaking vigorously. </li> <li>Add <font color=#357EC7>111 µl  of <font color=#357EC7>20 mg/ml lysozyme. Incubate on <font color=#357EC7><b><font color=#357EC7>ice  </b> for <font color=#357EC7>30 mins . </li> <li>Meanwhile: Measure out <font color=#357EC7>250 µl  of <font color=#357EC7>10% SDS into Eppendorf tube (3). Add <font color=#357EC7>125 µl  of <font color=#357EC7>4M NaOH. Add <font color=#357EC7>2.125 ml  of <font color=#357EC7>autoclaved water. Vortex the mixture for a few secs. </li> <li>Measure out <font color=#357EC7>2 ml  of <font color=#357EC7>SDS/NaOH mix into Oakridge tube (1). Incubate on <font color=#357EC7><b><font color=#357EC7>ice  </b> for <font color=#357EC7>10 mins . </li> <li>Add <font color=#357EC7>1.5 ml  of <a href="#Solution 3" ><font color=#357EC7>Solution 3 </a>. Incubate on <font color=#357EC7><b><font color=#357EC7>ice  </b> for <font color=#357EC7>10 mins . </li> <li>Vortex the mixture for a few secs. Centrifuge at a speed of <font color=#357EC7>17200 Xg for <font color=#357EC7>15 mins  at <font color=#357EC7>4°C  and aspirate out the top layer. Transfer top aqueous layer into Oakridge tube (2). Discard bottom layer. </li> <li>Measure out <font color=#357EC7>2.7 ml  of <font color=#357EC7>isopropanol into Oakridge tube (2). Centrifuge at a speed of <font color=#357EC7>17200 Xg 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% ethanol. Vortex the mixture for a few secs. Centrifuge at a speed of <font color=#357EC7>17200 Xg for <font color=#357EC7>10 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. Dry the pellet in air for <font color=#357EC7>2 - 5 mins . Add <font color=#357EC7>500 µl  of <font color=#357EC7>TE buffer. Resuspend pellet by vortexing/by shaking vigorously. Add <font color=#357EC7>500 µl  of <font color=#357EC7>5M LiCl. Incubate on <font color=#357EC7><b><font color=#357EC7>ice  </b> for <font color=#357EC7>5 mins . Centrifuge at a speed of <font color=#357EC7>17200 Xg for <font color=#357EC7>10 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> and aspirate out the top layer. Transfer top aqueous layer into Eppendorf tube (4). Discard bottom layer. </li> <li>Measure out <font color=#357EC7>1 ml  of <font color=#357EC7>isopropanol into Eppendorf tube (4). Incubate at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> for <font color=#357EC7>10 mins . </li> <li>Centrifuge at a speed of <font color=#357EC7>17200 Xg 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>100 µl  of <font color=#357EC7>70% ethanol. Vortex the mixture for a few secs. Centrifuge at a speed of <font color=#357EC7>17200 Xg for <font color=#357EC7>10 mins  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. Add <font color=#357EC7>375 µl  of <font color=#357EC7>TE buffer. Resuspend pellet by vortexing/by shaking vigorously. Add <font color=#357EC7>7.5 µl  of <font color=#357EC7>1 mg/ml RNaseA. Incubate at <font color=#357EC7>37°C  for <font color=#357EC7>30 mins . </li> <li>Add <font color=#357EC7>700 µl  of <a href="#phenol: chloroform: isoamyl alcohol" ><font color=#357EC7>phenol: chloroform: isoamyl alcohol </a>. Vortex the mixture for a few secs. <font color = "#800517">The solution should be thoroughly mixed. Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>2 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> and aspirate out the top layer. Transfer top aqueous layer into Eppendorf tube (5). Discard bottom layer. </li> <li>Repeat protocol from Step 14. Repeat until the interface between the phases is clear after centrifugation. </li> <li>Measure out <font color=#357EC7>700 µl  of <a href="#chloroform: isoamyl alcohol" ><font color=#357EC7>chloroform: isoamyl alcohol </a> into Eppendorf tube (5). Vortex the mixture for a few secs. <font color = "#800517">The solution should be thoroughly mixed. Centrifuge at <font color=#357EC7>maximum speed for <font color=#357EC7>2 mins  at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> and aspirate out the top layer. Transfer top aqueous layer into Eppendorf tube (6). Discard bottom layer. </li> <li>Repeat Step 16. <font color = "#800517">This removes phenol. </li> <li>Measure out <font color=#357EC7>750 µl  of <font color=#357EC7>straight ethanol into Eppendorf tube (6). Add <font color=#357EC7>125 µl  of <font color=#357EC7>3M sodium acetate. Option 1: Store at <font color=#357EC7>-80°C  for <font color=#357EC7>30 mins . (or) Option 2: Store at <font color=#357EC7>-20°C  for <font color=#357EC7>12 hrs (overnight). </li> <li>Centrifuge at a speed of <font color=#357EC7>13600 Xg for <font color=#357EC7>15 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. Add <font color=#357EC7>~100 µl  of <font color=#357EC7>70% ethanol. Vortex the mixture for a few secs. Centrifuge at a speed of <font color=#357EC7>13600 Xg for <font color=#357EC7>5 mins  at <font color=#357EC7>4°C , gently aspirate out the supernatant and discard it. Add <font color=#357EC7>100 - 200 µl  of <font color=#357EC7>TE buffer. Resuspend pellet by vortexing/by shaking vigorously. </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 15 hrs, 44 mins