Erman's Lab:DNA Miniprep with Alkaline Lysis protocol

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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;">...)
Current revision (02:48, 20 November 2009) (view source)
 
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<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;">(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>Flasks of appropriate volumes</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Inoculate <font color=#357EC7>2 ml of 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 Alkaline Lysis SLN1 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)<p><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></html>
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<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>

Current revision

Solutions/reagents:

Equipment:

  • Incubator
  • Centrifuge
  • Eppendorf tubes

Steps:

  1. Inoculate 2 ml LB (+ antibiotics) with single colony and incubate with shaking for 12 hrs(overnight) at 37°C.
  2. Measure out 2 ml of culture into Eppendorf tube (1).
  3. Centrifuge at maximum speed for 1.5 mins at 4°C, gently aspirate out the supernatant and discard it.
  4. Leave pellet as dry as possible.
  5. Add 100 µl of Alkaline Lysis SLN1.
    Resuspend pellet by vortexing/by shaking vigorously.
  6. Add 200 µl of freshly prepared AL2.
    Close the tube tightly and invert the tube 5 - 6 times.
    Do not vortex!
  7. Store the tube on ice.
  8. Add 300 µl of AL3.
    Close the tube tightly and gently mix the contents by inverting the tube.
  9. Incubate on ice for 5 mins.
  10. 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.
  11. Measure out 900 µl of isopropanol into Eppendorf tube (2).
    Vortex the mixture for a few secs.
  12. Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
  13. Add 1 ml of 70% EtOH.
    Vortex the mixture for a few secs.
  14. Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
  15. Dry the pellet in air.
  16. 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

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