Small Scale Plasmid Isolation (Miniprep) 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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<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>Sterile 1.5-ml microcentrifuge 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>1.5 ml</font></b> of <font color=#357EC7>overnight culture</font> into sterile 1.5-ml microcentrifuge tube (1).<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>1.5 ml</font></b> of <font color=#357EC7>overnight culture</font>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>200 µl</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>150 µl</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 1.5-ml microcentrifuge tube (2).<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 1.5-ml microcentrifuge tube (2).<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 1.5-ml microcentrifuge tube (3).<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 1.5-ml microcentrifuge tube (3).<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 1.5-ml microcentrifuge tube (4).<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 1.5-ml microcentrifuge tube (4).<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>200 µl</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>30 - 50 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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<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>Sterile 1.5-ml microcentrifuge 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>1.5 ml</font></b> of <font color=#357EC7>overnight culture</font> into sterile 1.5-ml microcentrifuge tube (1).<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>1.5 ml</font></b> of <font color=#357EC7>overnight culture</font>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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>200 µl</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>150 µl</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 1.5-ml microcentrifuge tube (2).<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 1.5-ml microcentrifuge tube (2).<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 1.5-ml microcentrifuge tube (3).<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 1.5-ml microcentrifuge tube (3).<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 1.5-ml microcentrifuge tube (4).<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 1.5-ml microcentrifuge tube (4).<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>200 µl</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>30 - 50 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, 21 mins</font></b></p>
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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
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Cell Lysis

    1. Measure out 1.5 ml of overnight culture into sterile 1.5-ml microcentrifuge tube (1).
      Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it.
    2. Add 1.5 ml of overnight culture.
      Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it.
    3. Add 1 ml of ice-cold Solution I.
      Resuspend pellet by vortexing/by shaking vigorously.
    4. Add 200 µl of Solution II.
      Close the tube tightly and gently mix the contents by inverting the tube.
      Store the tube on ice for 5 mins.
    5. Add 150 µl of Solution III.
      Vortex the mixture for a few secs.
      Store the tube on ice for 10 mins.
    6. Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (2).
      Discard bottom layer.
  2. Phenol/Chloroform Cleanup

    1. Add 0.5 volume phenol to sterile 1.5-ml microcentrifuge tube (2).
      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 1.5-ml microcentrifuge tube (3).
      Discard bottom layer.
  3. RNase (Optional)

    1. Measure out RNase to a final concentration of 3µg/ml into sterile 1.5-ml microcentrifuge tube (3).
    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 1.5-ml microcentrifuge tube (4).
      Discard bottom layer.

  4. Alcohol Precipitation / Purification

    1. Add 1 volume ice-cold isopropanol to sterile 1.5-ml microcentrifuge tube (4).
    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 200 µl 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 30 - 50 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, 21 mins

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