Erman's Lab:DNA Miniprep with Alkaline Lysis protocol: Difference between revisions
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New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li>2 ml of LB (+ antibiotics)</li><li> <a name="Alkaline Lysis SLN1">Alkaline Lysis SLN1 <i><br><tab><div style="margin-right: 600px;">... |
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> | <html><h2>Solutions/reagents:</h2><ul type="circle"><li>LB (+ antibiotics)</li><li> <a name="Alkaline Lysis SLN1">Alkaline Lysis SLN1 <i><br><tab><div style="margin-right: 600px;">(20mM Tris(pH 8), 50mM Glucose, 10mM EDTA)</div></i></a></li><li> <a name="freshly prepared AL2">freshly prepared AL2 <i><br><tab><div style="margin-right: 600px;">(0.2N NaOH, 1% SDS)</div></i></a></li><li> <a name="AL3">AL3 <i><br><tab><div style="margin-right: 600px;">(3M KAc, glacial acetic acid)</div></i></a></li><li>isopropanol stored at room temperature</li><li>70% EtOH</li><li>TE</li><li>water</li><li>single colony</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Inoculate <font color=#357EC7>2 ml LB (+ antibiotics)</font> with <font color=#357EC7>single colony</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>2 ml</font></b> of <font color=#357EC7>culture</font> into Eppendorf tube (1).<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>1.5 mins</font></b> at <b><font color=#357EC7>4°C</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li><font color = "#800517"><i>Leave pellet as dry as possible.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>100 µl</font></b> of <a href="#Alkaline Lysis SLN1" ><font color=#357EC7>Alkaline Lysis SLN1</font></a>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <a href="#freshly prepared AL2" ><font color=#357EC7>freshly prepared AL2</font></a>.<br>Close the tube tightly and invert the tube <b><font color=#357EC7>5 - 6 times</font></b>.<br><font color = "#800517"><i>Do not vortex!</i></font><br></li></p><p><li>Store the tube <b><font color=#357EC7>on ice</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>300 µl</font></b> of <a href="#AL3" ><font color=#357EC7>AL3</font></a>.<br>Close the tube tightly and gently mix the contents by inverting the tube.<br></li></p><p><li>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></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7>4°C</font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (2).<br>Discard bottom layer.<br></li></p><p><li>Measure out <b><font color=#357EC7>900 µl</font></b> of <font color=#357EC7>isopropanol</font> into Eppendorf tube (2).<br>Vortex the mixture for a few secs.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</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% EtOH</font>.<br>Vortex the mixture for a few secs.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 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>Dry the pellet in air. <br></li></p><p><li><p><b>Option 1: </b>Add <b><font color=#357EC7>50 µl</font></b> of <font color=#357EC7>TE</font>.<br>(or)<br><b>Option 2: </b>Add <b><font color=#357EC7>50 µl</font></b> of <font color=#357EC7>water</font>.<br></p><p>Dissolve the pellet in the solution.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 12 hrs, 26 mins</font></b></p></html> | ||
Latest revision as of 06:48, 20 November 2009
<html>
Solutions/reagents:
- LB (+ antibiotics)
- <a name="Alkaline Lysis SLN1">Alkaline Lysis SLN1
<tab>(20mM Tris(pH 8), 50mM Glucose, 10mM EDTA)</a> - <a name="freshly prepared AL2">freshly prepared AL2
<tab>(0.2N NaOH, 1% SDS)</a> - <a name="AL3">AL3
<tab>(3M KAc, glacial acetic acid)</a> - isopropanol stored at room temperature
- 70% EtOH
- TE
- water
- single colony
Equipment:
- Incubator
- Centrifuge
- Eppendorf tubes
Steps:
- Inoculate 2 ml LB (+ antibiotics) with single colony and incubate with shaking for 12 hrs(overnight) at 37°C.
- Measure out 2 ml of culture into Eppendorf tube (1).
- Centrifuge at maximum speed for 1.5 mins at 4°C, gently aspirate out the supernatant and discard it.
- Leave pellet as dry as possible.
- Add 100 µl of <a href="#Alkaline Lysis SLN1" >Alkaline Lysis SLN1</a>.
Resuspend pellet by vortexing/by shaking vigorously. - Add 200 µl of <a href="#freshly prepared AL2" >freshly prepared AL2</a>.
Close the tube tightly and invert the tube 5 - 6 times.
Do not vortex! - Store the tube on ice.
- Add 300 µl of <a href="#AL3" >AL3</a>.
Close the tube tightly and gently mix the contents by inverting the tube. - Incubate on ice for 5 mins.
- Centrifuge at maximum speed for 5 mins at 4°C and aspirate out the top layer.
Transfer top aqueous layer into Eppendorf tube (2).
Discard bottom layer. - Measure out 900 µl of isopropanol into Eppendorf tube (2).
Vortex the mixture for a few secs. - Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
- Add 1 ml of 70% EtOH.
Vortex the mixture for a few secs. - Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
- Dry the pellet in air.
Option 1: Add 50 µl of TE.
(or)
Option 2: Add 50 µl of water.Dissolve the pellet in the solution.
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 12 hrs, 26 mins
</html>
