Maxiprep of plasmid DNA from E.coli protocol

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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>LB broth + selective marker</li><li>50% sterile glycerol</li><li> <a name="TEG">TEG <tab><div style="margin-right: 600px;">(25mM Tris-Cl, 10mM EDTA, 50mM dextrose)</div></a></li><li>20 mg/ml lysozyme</li><li>10% SDS</li><li>4M NaOH</li><li>autoclaved water</li><li> <a name="Solution 3">Solution 3 <tab><div style="margin-right: 600px;">(3M potassium-acetate, 2M acetic acid -- glacial is 17M)</div></a></li><li>isopropanol</li><li>70% ethanol</li><li>TE buffer</li><li>5M LiCl</li><li>1 mg/ml RNaseA</li><li> <a name="phenol: chloroform: isoamyl alcohol">phenol: chloroform: isoamyl alcohol <tab><div style="margin-right: 600px;">(25:24:1)</div></a></li><li> <a name="chloroform: isoamyl alcohol">chloroform: isoamyl alcohol <tab><div style="margin-right: 600px;">(24:1)</div></a></li><li>straight ethanol</li><li>3M sodium acetate</li><li>a single colony of E. coli</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Eppendorf tubes</li><li>Oakridge tubes</li></ul><h2>Steps:</h2><ol><li>Inoculate 50 ml LB broth + selective marker with a single colony of E. coli and incubate with shaking for 12 hrs(overnight) at 37°C. </li><li>Measure out 850 µl of culture into Eppendorf tube (1). Add 150 µl of 50% sterile glycerol. Store at -80°C. Measure out 850 µl of culture into Eppendorf tube (2). Add 150 µl of 50% sterile glycerol. Store at -80°C. </li><li>Measure out as much culture as will fit into Oakridge tube (1). Centrifuge at a speed of 5800 Xg for 10 mins at 4°C, gently aspirate out the supernatant and discard it. Add rest of the culture to pellet. Centrifuge at a speed of 5800 Xg for 10 mins at 4°C, gently aspirate out the supernatant and discard it. Add 1 ml of <a href="#TEG" >TEG</a>. Resuspend pellet by vortexing/by shaking vigorously. </li><li>Add 111 µl of 20 mg/ml lysozyme. Incubate on ice for 30 mins. </li><li>Meanwhile: Measure out 250 µl of 10% SDS into Eppendorf tube (3). Add 125 µl of 4M NaOH. Add 2.125 ml of autoclaved water. Vortex the mixture for a few secs. </li><li>Measure out 2 ml of SDS/NaOH mix into Oakridge tube (1). Incubate on ice for 10 mins. </li><li>Add 1.5 ml of <a href="#Solution 3" >Solution 3</a>. Incubate on ice for 10 mins. </li><li>Vortex the mixture for a few secs. Centrifuge at a speed of 17200 Xg for 15 mins at 4°C and aspirate out the top layer. Transfer top aqueous layer into Oakridge tube (2). Discard bottom layer. </li><li>Measure out 2.7 ml of isopropanol into Oakridge tube (2). Centrifuge at a speed of 17200 Xg for 10 mins at room temperature, gently aspirate out the supernatant and discard it. </li><li>Add 1 ml of 70% ethanol. Vortex the mixture for a few secs. Centrifuge at a speed of 17200 Xg for 10 mins at room temperature, gently aspirate out the supernatant and discard it. Dry the pellet in air for 2 - 5 mins. Add 500 µl of TE buffer. Resuspend pellet by vortexing/by shaking vigorously. Add 500 µl of 5M LiCl. Incubate on ice for 5 mins. Centrifuge at a speed of 17200 Xg for 10 mins at room temperature and aspirate out the top layer. Transfer top aqueous layer into Eppendorf tube (4). Discard bottom layer. </li><li>Measure out 1 ml of isopropanol into Eppendorf tube (4). Incubate at room temperature for 10 mins. </li><li>Centrifuge at a speed of 17200 Xg for 10 mins at room temperature, gently aspirate out the supernatant and discard it. </li><li>Add 100 µl of 70% ethanol. Vortex the mixture for a few secs. Centrifuge at a speed of 17200 Xg for 10 mins at room temperature, gently aspirate out the supernatant and discard it. Add 375 µl of TE buffer. Resuspend pellet by vortexing/by shaking vigorously. Add 7.5 µl of 1 mg/ml RNaseA. Incubate at 37°C for 30 mins. </li><li>Add 700 µl of <a href="#phenol: chloroform: isoamyl alcohol" >phenol: chloroform: isoamyl alcohol</a>. Vortex the mixture for a few secs. The solution should be thoroughly mixed. Centrifuge at maximum speed for 2 mins at room temperature and aspirate out the top layer. Transfer top aqueous layer into Eppendorf tube (5). Discard bottom layer. </li><li>Repeat protocol from Step 14 until the interface between the phases is clear after centrifugation. </li><li>Measure out 700 µl of <a href="#chloroform: isoamyl alcohol" >chloroform: isoamyl alcohol</a> into Eppendorf tube (5). Vortex the mixture for a few secs. The solution should be thoroughly mixed. Centrifuge at maximum speed for 2 mins at room temperature and aspirate out the top layer. Transfer top aqueous layer into Eppendorf tube (6). Discard bottom layer. </li><li>Repeat Step 16. This removes phenol. </li><li>Measure out 750 µl of straight ethanol into Eppendorf tube (6). Add 125 µl of 3M sodium acetate. Option 1: Store at -80°C for 30 mins. (or) Option 2: Store at -20°C for 12 hrs(overnight). </li><li>Centrifuge at a speed of 13600 Xg for 15 mins at 4°C, gently aspirate out the supernatant and discard it. Add ~100 µl of 70% ethanol. Vortex the mixture for a few secs. Centrifuge at a speed of 13600 Xg for 5 mins at 4°C, gently aspirate out the supernatant and discard it. Add 100 - 200 µl of TE buffer. Resuspend pellet by vortexing/by shaking vigorously. </li></ol>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 15 hrs, 44 mins</html>

Maxiprep of plasmid DNA from E.coli protocol

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