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...)
Current revision (02:11, 20 November 2009) (view source)
 
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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>
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<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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Current revision

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

    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. Add 1 ml of ice-cold Solution I.
      Resuspend pellet by vortexing/by shaking vigorously.
    3. 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.
    4. Add 1.5 ml of Solution III.
      Vortex the mixture for a few secs.
      Store the tube on ice for 10 mins.
    5. Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 15-ml centrifuge tube (1).
      Discard bottom layer.
  2. Phenol/Chloroform Cleanup

    1. Add 0.5 volume phenol to sterile 15-ml centrifuge tube (1).
      Add 0.5 volume chloroform.
    2. Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 15-ml centrifuge tube (2).
      Discard bottom layer.
  3. RNase (Optional)

    1. Measure out RNase to a final concentration of 3µg/ml into sterile 15-ml centrifuge tube (2).
    2. Incubate at 37°C for 30 - 45 mins.
      Use a water bath.
    3. 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 room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 15-ml centrifuge tube (3).
      Discard bottom layer.

  4. Alcohol Precipitation / Purification

    1. Add 1 volume ice-cold isopropanol to sterile 15-ml centrifuge tube (3).
    2. Close the tube tightly and gently mix the contents by inverting the tube.
      Store the tube on ice for 10 mins.
    3. Centrifuge at maximum speed for 15 mins at room temperature, gently aspirate out the supernatant and discard it.
    4. Add 2 ml of 70% ethanol.
      Mix solution by pipetting up and down several times.
    5. Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
    6. Dry the pellet in air for at most 30 mins.
    7. Add 200 - 300 µl of sterile distilled water.
      Resuspend pellet by vortexing/by shaking vigorously.
    8. Store at -20°C.

    TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 2 hrs, 20 mins

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