Difference between revisions of "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 <i><br><tab><div style="margin-right: 600px;">(25mM Tris-Cl, 10mM EDTA, 50mM dextrose)</div></i></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 <i><br><tab><div style="margin-right: 600px;">(3M potassium-acetate, 2M acetic acid -- glacial is 17M)</div></i></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 <i><br><tab><div style="margin-right: 600px;">(25:24:1)</div></i></a></li><li> <a name="chloroform: isoamyl alcohol">chloroform: isoamyl alcohol <i><br><tab><div style="margin-right: 600px;">(24:1)</div></i></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><p><li>Inoculate <font color=#357EC7>50 ml LB broth + selective marker</font> with <font color=#357EC7>a single colony of E. coli</font> and incubate with shaking for <b><font color=#357EC7>12 hrs</font></b>(overnight) at <b><font color=#357EC7>37°C</font></b>.<br></li></p><p><li>Measure out <b><font color=#357EC7>850 µl</font></b> of <font color=#357EC7>culture</font> into Eppendorf tube (1).<br>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>50% sterile glycerol</font>.<br>Store at <b><font color=#357EC7>-80°C</font></b>.<br>Measure out <b><font color=#357EC7>850 µl</font></b> of <font color=#357EC7>culture</font> into Eppendorf tube (2).<br>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>50% sterile glycerol</font>.<br>Store at <b><font color=#357EC7>-80°C</font></b>.<br></li></p><p><li>Measure out as much culture as will fit into Oakridge tube (1).<br>Centrifuge at a speed of <font color=#357EC7>5800 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <font color=#357EC7>rest of the culture</font> to pellet.<br>Centrifuge at a speed of <font color=#357EC7>5800 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>1 ml</font></b> of <a href="#TEG" ><font color=#357EC7>TEG</font></a>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>111 µl</font></b> of <font color=#357EC7>20 mg/ml lysozyme</font>.<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li><b>Meanwhile:</b><br>Measure out <b><font color=#357EC7>250 µl</font></b> of <font color=#357EC7>10% SDS</font> into Eppendorf tube (3).<br>Add <b><font color=#357EC7>125 µl</font></b> of <font color=#357EC7>4M NaOH</font>.<br>Add <b><font color=#357EC7>2.125 ml</font></b> of <font color=#357EC7>autoclaved water</font>.<br>Vortex the mixture for a few secs.<br></li></p><p><li>Measure out <b><font color=#357EC7>2 ml</font></b> of <font color=#357EC7>SDS/NaOH mix</font> into Oakridge tube (1).<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>1.5 ml</font></b> of <a href="#Solution 3" ><font color=#357EC7>Solution 3</font></a>.<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>15 mins</font></b> at <b><font color=#357EC7>4°C</font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Oakridge tube (2).<br>Discard bottom layer.<br></li></p><p><li>Measure out <b><font color=#357EC7>2.7 ml</font></b> of <font color=#357EC7>isopropanol</font> into Oakridge tube (2).<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Dry the pellet in air for <b><font color=#357EC7>2 - 5 mins</font></b>.<br>Add <b><font color=#357EC7>500 µl</font></b> of <font color=#357EC7>TE buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br>Add <b><font color=#357EC7>500 µl</font></b> of <font color=#357EC7>5M LiCl</font>.<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>5 mins</font></b>.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (4).<br>Discard bottom layer.<br></li></p><p><li>Measure out <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>isopropanol</font> into Eppendorf tube (4).<br>Incubate at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>100 µl</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>375 µl</font></b> of <font color=#357EC7>TE buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br>Add <b><font color=#357EC7>7.5 µl</font></b> of <font color=#357EC7>1 mg/ml RNaseA</font>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>700 µl</font></b> of <a href="#phenol: chloroform: isoamyl alcohol" ><font color=#357EC7>phenol: chloroform: isoamyl alcohol</font></a>.<br>Vortex the mixture for a few secs.<br><font color = "#800517"><i>The solution should be thoroughly mixed.</i></font><br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (5).<br>Discard bottom layer.<br></li></p><p><li>Repeat protocol from Step 14 until the interface between the phases is clear after centrifugation.<br></li></p><p><li>Measure out <b><font color=#357EC7>700 µl</font></b> of <a href="#chloroform: isoamyl alcohol" ><font color=#357EC7>chloroform: isoamyl alcohol</font></a> into Eppendorf tube (5).<br>Vortex the mixture for a few secs.<br><font color = "#800517"><i>The solution should be thoroughly mixed.</i></font><br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (6).<br>Discard bottom layer.<br></li></p><p><li>Repeat Step 16. <br><font color = "#800517"><i>This removes phenol.</i></font><br></li></p><p><li>Measure out <b><font color=#357EC7>750 µl</font></b> of <font color=#357EC7>straight ethanol</font> into Eppendorf tube (6).<br>Add <b><font color=#357EC7>125 µl</font></b> of <font color=#357EC7>3M sodium acetate</font>.<br><p><b>Option 1: </b>Store at <b><font color=#357EC7>-80°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br>(or)<br><b>Option 2: </b>Store at <b><font color=#357EC7>-20°C</font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></p><p></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>13600 Xg</font> for <b><font color=#357EC7>15 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>~100 µl</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>13600 Xg</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>100 - 200 µl</font></b> of <font color=#357EC7>TE buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 15 hrs, 44 mins</font></b></p></html>
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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 <i><br><tab><div style="margin-right: 600px;">(25mM Tris-Cl, 10mM EDTA, 50mM dextrose)</div></i></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 <i><br><tab><div style="margin-right: 600px;">(3M potassium-acetate, 2M acetic acid -- glacial is 17M)</div></i></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 <i><br><tab><div style="margin-right: 600px;">(25:24:1)</div></i></a></li><li> <a name="chloroform: isoamyl alcohol">chloroform: isoamyl alcohol <i><br><tab><div style="margin-right: 600px;">(24:1)</div></i></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><p><li>Inoculate <font color=#357EC7>50 ml LB broth + selective marker</font> with <font color=#357EC7>a single colony of E. coli</font> and incubate with shaking for <b><font color=#357EC7>12 hrs</font></b>(overnight) at <b><font color=#357EC7>37°C</font></b>.<br></li></p><p><li>Measure out <b><font color=#357EC7>850 µl</font></b> of <font color=#357EC7>culture</font> into Eppendorf tube (1).<br>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>50% sterile glycerol</font>.<br>Store at <b><font color=#357EC7>-80°C</font></b>.<br>Measure out <b><font color=#357EC7>850 µl</font></b> of <font color=#357EC7>culture</font> into Eppendorf tube (2).<br>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>50% sterile glycerol</font>.<br>Store at <b><font color=#357EC7>-80°C</font></b>.<br></li></p><p><li>Measure out as much culture as will fit into Oakridge tube (1).<br>Centrifuge at a speed of <font color=#357EC7>5800 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <font color=#357EC7>rest of the culture</font> to pellet.<br>Centrifuge at a speed of <font color=#357EC7>5800 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>1 ml</font></b> of <a href="#TEG" ><font color=#357EC7>TEG</font></a>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>111 µl</font></b> of <font color=#357EC7>20 mg/ml lysozyme</font>.<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li><b>Meanwhile:</b><br>Measure out <b><font color=#357EC7>250 µl</font></b> of <font color=#357EC7>10% SDS</font> into Eppendorf tube (3).<br>Add <b><font color=#357EC7>125 µl</font></b> of <font color=#357EC7>4M NaOH</font>.<br>Add <b><font color=#357EC7>2.125 ml</font></b> of <font color=#357EC7>autoclaved water</font>.<br>Vortex the mixture for a few secs.<br></li></p><p><li>Measure out <b><font color=#357EC7>2 ml</font></b> of <font color=#357EC7>SDS/NaOH mix</font> into Oakridge tube (1).<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>1.5 ml</font></b> of <a href="#Solution 3" ><font color=#357EC7>Solution 3</font></a>.<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>15 mins</font></b> at <b><font color=#357EC7>4°C</font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Oakridge tube (2).<br>Discard bottom layer.<br></li></p><p><li>Measure out <b><font color=#357EC7>2.7 ml</font></b> of <font color=#357EC7>isopropanol</font> into Oakridge tube (2).<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Dry the pellet in air for <b><font color=#357EC7>2 - 5 mins</font></b>.<br>Add <b><font color=#357EC7>500 µl</font></b> of <font color=#357EC7>TE buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br>Add <b><font color=#357EC7>500 µl</font></b> of <font color=#357EC7>5M LiCl</font>.<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>5 mins</font></b>.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (4).<br>Discard bottom layer.<br></li></p><p><li>Measure out <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>isopropanol</font> into Eppendorf tube (4).<br>Incubate at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>100 µl</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>375 µl</font></b> of <font color=#357EC7>TE buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br>Add <b><font color=#357EC7>7.5 µl</font></b> of <font color=#357EC7>1 mg/ml RNaseA</font>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>700 µl</font></b> of <a href="#phenol: chloroform: isoamyl alcohol" ><font color=#357EC7>phenol: chloroform: isoamyl alcohol</font></a>.<br>Vortex the mixture for a few secs.<br><font color = "#800517"><i>The solution should be thoroughly mixed.</i></font><br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (5).<br>Discard bottom layer.<br></li></p><p><li>Repeat protocol from Step 14.<br><font color=red>Repeat until the interface between the phases is clear after centrifugation.</font><br></li></p><p><li>Measure out <b><font color=#357EC7>700 µl</font></b> of <a href="#chloroform: isoamyl alcohol" ><font color=#357EC7>chloroform: isoamyl alcohol</font></a> into Eppendorf tube (5).<br>Vortex the mixture for a few secs.<br><font color = "#800517"><i>The solution should be thoroughly mixed.</i></font><br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (6).<br>Discard bottom layer.<br></li></p><p><li>Repeat Step 16. <br><font color = "#800517"><i>This removes phenol.</i></font><br></li></p><p><li>Measure out <b><font color=#357EC7>750 µl</font></b> of <font color=#357EC7>straight ethanol</font> into Eppendorf tube (6).<br>Add <b><font color=#357EC7>125 µl</font></b> of <font color=#357EC7>3M sodium acetate</font>.<br><p><b>Option 1: </b>Store at <b><font color=#357EC7>-80°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br>(or)<br><b>Option 2: </b>Store at <b><font color=#357EC7>-20°C</font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></p><p></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>13600 Xg</font> for <b><font color=#357EC7>15 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>~100 µl</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>13600 Xg</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>100 - 200 µl</font></b> of <font color=#357EC7>TE buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 15 hrs, 44 mins</font></b></p></html>

Latest revision as of 01:22, 9 January 2010

<html><h2>Solutions/reagents:</h2><ul type="circle"><li>LB broth + selective marker</li><li>50% sterile glycerol</li><li> <a name="TEG">TEG <i><br><tab><div style="margin-right: 600px;">(25mM Tris-Cl, 10mM EDTA, 50mM dextrose)</div></i></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 <i><br><tab><div style="margin-right: 600px;">(3M potassium-acetate, 2M acetic acid -- glacial is 17M)</div></i></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 <i><br><tab><div style="margin-right: 600px;">(25:24:1)</div></i></a></li><li> <a name="chloroform: isoamyl alcohol">chloroform: isoamyl alcohol <i><br><tab><div style="margin-right: 600px;">(24:1)</div></i></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><p><li>Inoculate <font color=#357EC7>50 ml LB broth + selective marker</font> with <font color=#357EC7>a single colony of E. coli</font> and incubate with shaking for <b><font color=#357EC7>12 hrs</font></b>(overnight) at <b><font color=#357EC7>37°C</font></b>.<br></li></p><p><li>Measure out <b><font color=#357EC7>850 µl</font></b> of <font color=#357EC7>culture</font> into Eppendorf tube (1).<br>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>50% sterile glycerol</font>.<br>Store at <b><font color=#357EC7>-80°C</font></b>.<br>Measure out <b><font color=#357EC7>850 µl</font></b> of <font color=#357EC7>culture</font> into Eppendorf tube (2).<br>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>50% sterile glycerol</font>.<br>Store at <b><font color=#357EC7>-80°C</font></b>.<br></li></p><p><li>Measure out as much culture as will fit into Oakridge tube (1).<br>Centrifuge at a speed of <font color=#357EC7>5800 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <font color=#357EC7>rest of the culture</font> to pellet.<br>Centrifuge at a speed of <font color=#357EC7>5800 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>1 ml</font></b> of <a href="#TEG" ><font color=#357EC7>TEG</font></a>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>111 µl</font></b> of <font color=#357EC7>20 mg/ml lysozyme</font>.<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li><b>Meanwhile:</b><br>Measure out <b><font color=#357EC7>250 µl</font></b> of <font color=#357EC7>10% SDS</font> into Eppendorf tube (3).<br>Add <b><font color=#357EC7>125 µl</font></b> of <font color=#357EC7>4M NaOH</font>.<br>Add <b><font color=#357EC7>2.125 ml</font></b> of <font color=#357EC7>autoclaved water</font>.<br>Vortex the mixture for a few secs.<br></li></p><p><li>Measure out <b><font color=#357EC7>2 ml</font></b> of <font color=#357EC7>SDS/NaOH mix</font> into Oakridge tube (1).<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>1.5 ml</font></b> of <a href="#Solution 3" ><font color=#357EC7>Solution 3</font></a>.<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>15 mins</font></b> at <b><font color=#357EC7>4°C</font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Oakridge tube (2).<br>Discard bottom layer.<br></li></p><p><li>Measure out <b><font color=#357EC7>2.7 ml</font></b> of <font color=#357EC7>isopropanol</font> into Oakridge tube (2).<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Dry the pellet in air for <b><font color=#357EC7>2 - 5 mins</font></b>.<br>Add <b><font color=#357EC7>500 µl</font></b> of <font color=#357EC7>TE buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br>Add <b><font color=#357EC7>500 µl</font></b> of <font color=#357EC7>5M LiCl</font>.<br>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>5 mins</font></b>.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (4).<br>Discard bottom layer.<br></li></p><p><li>Measure out <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>isopropanol</font> into Eppendorf tube (4).<br>Incubate at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>10 mins</font></b>.<br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>100 µl</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>17200 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>375 µl</font></b> of <font color=#357EC7>TE buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br>Add <b><font color=#357EC7>7.5 µl</font></b> of <font color=#357EC7>1 mg/ml RNaseA</font>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>700 µl</font></b> of <a href="#phenol: chloroform: isoamyl alcohol" ><font color=#357EC7>phenol: chloroform: isoamyl alcohol</font></a>.<br>Vortex the mixture for a few secs.<br><font color = "#800517"><i>The solution should be thoroughly mixed.</i></font><br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (5).<br>Discard bottom layer.<br></li></p><p><li>Repeat protocol from Step 14.<br><font color=red>Repeat until the interface between the phases is clear after centrifugation.</font><br></li></p><p><li>Measure out <b><font color=#357EC7>700 µl</font></b> of <a href="#chloroform: isoamyl alcohol" ><font color=#357EC7>chloroform: isoamyl alcohol</font></a> into Eppendorf tube (5).<br>Vortex the mixture for a few secs.<br><font color = "#800517"><i>The solution should be thoroughly mixed.</i></font><br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (6).<br>Discard bottom layer.<br></li></p><p><li>Repeat Step 16. <br><font color = "#800517"><i>This removes phenol.</i></font><br></li></p><p><li>Measure out <b><font color=#357EC7>750 µl</font></b> of <font color=#357EC7>straight ethanol</font> into Eppendorf tube (6).<br>Add <b><font color=#357EC7>125 µl</font></b> of <font color=#357EC7>3M sodium acetate</font>.<br><p><b>Option 1: </b>Store at <b><font color=#357EC7>-80°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br>(or)<br><b>Option 2: </b>Store at <b><font color=#357EC7>-20°C</font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></p><p></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>13600 Xg</font> for <b><font color=#357EC7>15 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>~100 µl</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at a speed of <font color=#357EC7>13600 Xg</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>100 - 200 µl</font></b> of <font color=#357EC7>TE buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 15 hrs, 44 mins</font></b></p></html>