Chromosomal DNA isolation from E. coli protocol

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(New page: <html> <h2>Solutions/reagents:</h2><ul type="circle"><li>culture grown in your favorite medium</li><li>ice-cold Killing Buffer</li><li>TE</li><li>10% SDS</li><li>0.5M EDTA</li><li>isopropa...)
Current revision (02:51, 20 November 2009) (view source)
 
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<h2>Solutions/reagents:</h2><ul type="circle"><li>culture grown in your favorite medium</li><li>ice-cold Killing Buffer</li><li>TE</li><li>10% SDS</li><li>0.5M EDTA</li><li>isopropanol</li><li> <a name="RNase A">RNase A <i><br><tab><div style="margin-right: 600px;">(25mg/ml)</div></i></a></li><li> <a name="proteinase K">proteinase K <i><br><tab><div style="margin-right: 600px;">(25mg/ml)</div></i></a></li><li>ethanol</li><li>3M Na-Acetate</li><li>70% ethanol</li><li>distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out culture grown in your favorite medium into an Eppendorf tube.<br>Add  <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>ice-cold Killing Buffer</font>.<br>Vortex the mixture for a few secs.<br>Store the tube on ice.<br><font color = "#800517"><i>Samples should be processed as fast as possible.</i></font><br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>Add <b><font color=#357EC7>300 µl</font></b> of <font color=#357EC7>TE</font>.<br>Add <b><font color=#357EC7>40 µl</font></b> of <font color=#357EC7>10% SDS</font>.<br>Add <b><font color=#357EC7>3 µl</font></b> of <font color=#357EC7>0.5M EDTA</font>.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Incubate at <b><font color=#357EC7>65°C</font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>750 µl</font></b> of <font color=#357EC7>isopropanol</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>Add <b><font color=#357EC7>500 µl</font></b> of <font color=#357EC7>TE</font>.<br>Add <b><font color=#357EC7>2 µl</font></b> of <a href="#RNase A" ><font color=#357EC7>RNase A</font></a>.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Incubate at <b><font color=#357EC7>65°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>2 µl</font></b> of <a href="#proteinase K" ><font color=#357EC7>proteinase K</font></a>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li><font color = "red"><i>phenol extract (2x phenol & 2x chlorophorm).</i></font><br></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ethanol</font>.<br>Add <b><font color=#357EC7>40 µl</font></b> of <font color=#357EC7>3M Na-Acetate</font>.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</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>50 µl</font></b> of <font color=#357EC7>distilled water</font>.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p></ol></html>
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<h2>Solutions/reagents:</h2><ul type="circle"><li>culture grown in your favorite medium</li><li>ice-cold Killing Buffer</li><li>TE</li><li>10% SDS</li><li>0.5M EDTA</li><li>isopropanol</li><li> <a name="RNase A">RNase A <i><br><tab><div style="margin-right: 600px;">(25mg/ml)</div></i></a></li><li> <a name="proteinase K">proteinase K <i><br><tab><div style="margin-right: 600px;">(25mg/ml)</div></i></a></li><li>ethanol</li><li>3M Na-Acetate</li><li>distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>ice-cold Killing Buffer</font> into culture grown in your favorite medium.<br>Vortex the mixture for a few secs.<br>Store the tube <b><font color=#357EC7>on ice</font></b>.<br><font color = "#800517"><i>Samples should be processed as fast as possible.</i></font><br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>Measure out <b><font color=#357EC7>300 µl</font></b> of <font color=#357EC7>TE</font> into Eppendorf tube (1).<br>Add <b><font color=#357EC7>40 µl</font></b> of <font color=#357EC7>10% SDS</font>.<br>Add <b><font color=#357EC7>3 µl</font></b> of <font color=#357EC7>0.5M EDTA</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Incubate at <b><font color=#357EC7>65°C</font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>750 µl</font></b> of <font color=#357EC7>isopropanol</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>Add <b><font color=#357EC7>500 µl</font></b> of <font color=#357EC7>TE</font>.<br>Add <b><font color=#357EC7>2 µl</font></b> of <a href="#RNase A" ><font color=#357EC7>RNase A</font></a>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Incubate at <b><font color=#357EC7>65°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>2 µl</font></b> of <a href="#proteinase K" ><font color=#357EC7>proteinase K</font></a>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br></li></p><p><li><font color = "red"><i>phenol extract (2x phenol & 2x chlorophorm).</i></font><br></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>ethanol</font>.<br>Add <b><font color=#357EC7>40 µl</font></b> of <font color=#357EC7>3M Na-Acetate</font>.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</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>50 µl</font></b> of <font color=#357EC7>distilled water</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>~ 13 hrs, 13 mins</font></b></p></html>

Current revision

Solutions/reagents:

Equipment:

  • Centrifuge
  • Incubator

Steps:

  1. Measure out 1 volume ice-cold Killing Buffer into culture grown in your favorite medium.
    Vortex the mixture for a few secs.
    Store the tube on ice.
    Samples should be processed as fast as possible.
  2. Centrifuge at maximum speed for 3 mins at 4°C, gently aspirate out the supernatant and discard it.
  3. Measure out 300 µl of TE into Eppendorf tube (1).
    Add 40 µl of 10% SDS.
    Add 3 µl of 0.5M EDTA.
    Resuspend pellet by vortexing/by shaking vigorously.
  4. Incubate at 65°C for 5 mins.
  5. Add 750 µl of isopropanol.
    Vortex the mixture for a few secs.
  6. Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
  7. Add 500 µl of TE.
    Add 2 µl of RNase A.
    Resuspend pellet by vortexing/by shaking vigorously.
  8. Incubate at 65°C for 30 mins.
  9. Add 2 µl of proteinase K.
    Incubate at 37°C for 15 mins.
  10. phenol extract (2x phenol & 2x chlorophorm).
  11. Add 1 ml of ethanol.
    Add 40 µl of 3M Na-Acetate.
    Store at room temperature for 12 hrs(overnight).
  12. Centrifuge at maximum speed for 15 mins at 4°C, gently aspirate out the supernatant and discard it.
    Add 50 µl of distilled water.
    Resuspend pellet by vortexing/by shaking vigorously.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 13 mins

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