Difference between revisions of "Small Scale Plasmid Isolation (Maxiprep) protocol"

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(New page: <html> <h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li>ice-cold Solution I</li><li>Solution II</li><li>Solution III</li><li>phenol</li><li>chloroform</li><li>RN...)
 
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li>ice-cold Solution I</li><li>Solution II</li><li>Solution III</li><li>phenol</li><li>chloroform</li><li>RNase to a final concentration of 3µg/ml</li><li>ice-cold isopropanol</li><li>70% ethanol</li><li>sterile distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li><li>50-ml centrifuge tubes</li><li>Sterile 15-ml centrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Cell Lysis</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>50 ml</font></b> of <font color=#357EC7>overnight culture</font> into 50-ml centrifuge tube (1).<br>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>1 ml</font></b> of <font color=#357EC7>ice-cold Solution I</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>2 ml</font></b> of <font color=#357EC7>Solution II</font>.<br>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>Solution III</font>.<br>Vortex the mixture for a few secs.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 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><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into sterile 15-ml centrifuge tube (1).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Phenol/Chloroform Cleanup</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font> to sterile 15-ml centrifuge tube (1).<br>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br></li></p><p><li>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 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 sterile 15-ml centrifuge tube (2).<br>Discard bottom layer.<br></li></p></ol></li></p><p><b><font size=3>RNase (Optional)</font></b><br><ol type="a"><p><li>Measure out RNase to a final concentration of 3µg/ml into sterile 15-ml centrifuge tube (2).<br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>30 - 45 mins</font></b>.<br><font color = "#800517"><i>Use a water bath.</i></font><br></li></p><p><li>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font>.<br>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 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 sterile 15-ml centrifuge tube (3).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Alcohol Precipitation / Purification</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>ice-cold isopropanol</font> to sterile 15-ml centrifuge tube (3).<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 mins</font></b>.<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>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>2 ml</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Mix solution by pipetting up and down several times.<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 for at most <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>200 - 300 µl</font></b> of <font color=#357EC7>sterile distilled water</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p><p><li>Store at <b><font color=#357EC7>-20°C</font></b>.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 2 hrs, 20 mins</font></b></p>
<h2>Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li>ice-cold Solution I</li><li>Solution II</li><li>Solution III</li><li>phenol</li><li>chloroform</li><li>RNase to a final concentration of 3µg/ml</li><li>ice-cold isopropanol</li><li>70% ethanol</li><li>sterile distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li><li>Centrifuge bottles</li><li>Sterile 15-ml centrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Cell Lysis</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>50 ml</font></b> of <font color=#357EC7>overnight culture</font> into a centrifuge bottle.<br>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>1 ml</font></b> of <font color=#357EC7>ice-cold Solution I</font>.<br>Resuspend ice-cold Solution I by vortexing/by shaking vigorously.<br></li></p><p><li>Add <b><font color=#357EC7>2 ml</font></b> of <font color=#357EC7>Solution II</font>.<br>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>Solution III</font>.<br>Vortex the mixture for a few secs.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 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><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into sterile 15-ml centrifuge tube (1).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Phenol/Chloroform Cleanup</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font> to sterile 15-ml centrifuge tube (1).<br>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br></li></p><p><li>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 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 sterile 15-ml centrifuge tube (2).<br>Discard bottom layer.<br></li></p></ol></li></p><p><b><font size=3>RNase (Optional)</font></b><br><ol type="a"><p><li>Measure out RNase to a final concentration of 3µg/ml into sterile 15-ml centrifuge tube (2).<br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>30 - 45 mins</font></b>.<br><font color = "#800517"><i>Use a water bath.</i></font><br></li></p><p><li>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>phenol</font>.<br>Add  <b><font color=#357EC7>0.5</font></b> volume <font color=#357EC7>chloroform</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 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 sterile 15-ml centrifuge tube (3).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Alcohol Precipitation / Purification</font></b><br><ol type="a"><p><li>Add  <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>ice-cold isopropanol</font> to sterile 15-ml centrifuge tube (3).<br></li></p><p><li>Close the tube tightly and gently mix the contents by inverting the tube.<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 mins</font></b>.<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>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>2 ml</font></b> of <font color=#357EC7>70% ethanol</font>.<br>Mix solution by pipetting up and down several times.<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 for at most <b><font color=#357EC7>30 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>200 - 300 µl</font></b> of <font color=#357EC7>sterile distilled water</font>.<br>Resuspend sterile distilled water by vortexing/by shaking vigorously.<br></li></p><p><li>Store at <b><font color=#357EC7>-20°C</font></b>.<br></li></p></ol>
 
 
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Latest revision as of 00:11, 20 November 2009

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Solutions/reagents:

  • overnight culture
  • ice-cold Solution I
  • Solution II
  • Solution III
  • phenol
  • chloroform
  • RNase to a final concentration of 3µg/ml
  • ice-cold isopropanol
  • 70% ethanol
  • sterile distilled water

Equipment:

  • Centrifuge
  • Incubator
  • 50-ml centrifuge tubes
  • Sterile 15-ml centrifuge tubes

Steps:

  1. Cell Lysis
      <p>
    1. Measure out 50 ml of overnight culture into 50-ml centrifuge tube (1).
      Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
    2. </p><p>
    3. Add 1 ml of ice-cold Solution I.
      Resuspend pellet by vortexing/by shaking vigorously.
    4. </p><p>
    5. Add 2 ml of Solution II.
      Close the tube tightly and gently mix the contents by inverting the tube.
      Store the tube on ice for 5 mins.
    6. </p><p>
    7. Add 1.5 ml of Solution III.
      Vortex the mixture for a few secs.
      Store the tube on ice for 10 mins.
    8. </p><p>
    9. Centrifuge at maximum speed for 5 mins at <b>room temperature</b> and aspirate out the top layer.
      Transfer top aqueous layer into sterile 15-ml centrifuge tube (1).
      Discard bottom layer.
    10. </p>
  2. Phenol/Chloroform Cleanup
      <p>
    1. Add 0.5 volume phenol to sterile 15-ml centrifuge tube (1).
      Add 0.5 volume chloroform.
    2. </p><p>
    3. Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 5 mins at <b>room temperature</b> and aspirate out the top layer.
      Transfer top aqueous layer into sterile 15-ml centrifuge tube (2).
      Discard bottom layer.
    4. </p>
  3. RNase (Optional)

      <p>
    1. Measure out RNase to a final concentration of 3µg/ml into sterile 15-ml centrifuge tube (2).
    2. </p><p>
    3. Incubate at 37°C for 30 - 45 mins.
      Use a water bath.
    4. </p><p>
    5. Add 0.5 volume phenol.
      Add 0.5 volume chloroform.
      Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 5 mins at <b>room temperature</b> and aspirate out the top layer.
      Transfer top aqueous layer into sterile 15-ml centrifuge tube (3).
      Discard bottom layer.
    6. </p>
    </li>

  4. Alcohol Precipitation / Purification
      <p>
    1. Add 1 volume ice-cold isopropanol to sterile 15-ml centrifuge tube (3).
    2. </p><p>
    3. Close the tube tightly and gently mix the contents by inverting the tube.
      Store the tube on ice for 10 mins.
    4. </p><p>
    5. Centrifuge at maximum speed for 15 mins at room temperature, gently aspirate out the supernatant and discard it.
    6. </p><p>
    7. Add 2 ml of 70% ethanol.
      Mix solution by pipetting up and down several times.
    8. </p><p>
    9. Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
    10. </p><p>
    11. Dry the pellet in air for at most 30 mins.
    12. </p><p>
    13. Add 200 - 300 µl of sterile distilled water.
      Resuspend pellet by vortexing/by shaking vigorously.
    14. </p><p>
    15. Store at -20°C.
    16. </p>
    <p>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 2 hrs, 20 mins

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