Maxiprep of plasmid DNA from E.coli protocol: Difference between revisions

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Revision as of 06:25, 20 November 2009

<html>

Solutions/reagents:

LB broth + selective marker
50% sterile glycerol
<a name="TEG">TEG
<tab>
(25mM Tris-Cl, 10mM EDTA, 50mM dextrose)
</a>
20 mg/ml lysozyme
10% SDS
4M NaOH
autoclaved water
<a name="Solution 3">Solution 3
<tab>
(3M potassium-acetate, 2M acetic acid -- glacial is 17M)
</a>
isopropanol
70% ethanol
TE buffer
5M LiCl
1 mg/ml RNaseA
<a name="phenol: chloroform: isoamyl alcohol">phenol: chloroform: isoamyl alcohol
<tab>
(25:24:1)
</a>
<a name="chloroform: isoamyl alcohol">chloroform: isoamyl alcohol
<tab>
(24:1)
</a>
straight ethanol
3M sodium acetate
a single colony of E. coli

Equipment:

Incubator
Centrifuge
Eppendorf tubes
Oakridge tubes

Steps:

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.

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.

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.

Add 111 µl of 20 mg/ml lysozyme.
Incubate on ice for 30 mins.

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.

Measure out 2 ml of SDS/NaOH mix into Oakridge tube (1).
Incubate on ice for 10 mins.

Add 1.5 ml of <a href="#Solution 3" >Solution 3</a>.
Incubate on ice for 10 mins.

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.

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.

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.

Measure out 1 ml of isopropanol into Eppendorf tube (4).
Incubate at room temperature for 10 mins.

Centrifuge at a speed of 17200 Xg for 10 mins at room temperature, 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 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.

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.

Repeat protocol from Step 14 until the interface between the phases is clear after centrifugation.

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.

Repeat Step 16.
This removes phenol.

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).

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.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 15 hrs, 44 mins

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Revision as of 06:29, 23 September 2009 view source Vaishnavi Ananth (talk \| contribs) 278 edits New page: <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;">(2...		Revision as of 06:25, 20 November 2009 view source Vaishnavi Ananth (talk \| contribs) 278 edits No edit summary Newer edit →
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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>Flasks of appropriate volumes~~</li><li>Eppendorf tubes</li><li>Oakridge tubes</li></ul><h2>Steps:</h2><ol><p><li>Inoculate <font color=#357EC7>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~~>Set aside a fresh an Eppendorf tube. Call it Eppendorf 1. <br~~>Measure out <b><font color=#357EC7>850 µl</font></b> of <font color=#357EC7>culture</font> into Eppendorf 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>Set aside a fresh an Eppendorf tube. Call it Eppendorf 2~~. <br>Measure out <b><font color=#357EC7>850 µl</font></b> of <font color=#357EC7>culture</font> into Eppendorf 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 an Oakridge tube.<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 ~~the~~ 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 an Eppendorf tube.<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 µl</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 ~~the~~ Oakridge tube.<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 an Oakridge tube.<br>Discard bottom layer.<br></li></p><p><li>~~Add~~ <b><font color=#357EC7>2.7 ml</font></b> of <font color=#357EC7>isopropanol</font>.<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 ~~the~~ 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 an Eppendorf tube.<br>Discard bottom layer.<br></li></p><p><li>~~Add~~ <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>isopropanol</font>.<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 ~~the~~ 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 an Eppendorf tube.<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>~~Add~~ <b><font color=#357EC7>700 µl</font></b> of <a href="#chloroform: isoamyl alcohol" ><font color=#357EC7>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 an Eppendorf tube.<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>~~Add~~ <b><font color=#357EC7>750 µl</font></b> of <font color=#357EC7>straight ethanol</font>.<br>Add <b><font color=#357EC7>125 µl</font></b> of <font color=#357EC7>3M sodium acetate</font>.<br><p>Option 1: Store at <b><font color=#357EC7>-80°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br>(or)<br>Option 2: 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 ~~the~~ pellet by vortexing/by shaking vigorously.<br></li></p></ol></html>		<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>

Maxiprep of plasmid DNA from E.coli protocol: Difference between revisions

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