Kafatos:Minipreps protocol

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(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li>5 ml sterile LB medium (+ antibiotic)</li><li>P1 Buffer</li><li>P2 Buffer</li><li>ice-cold P3 Buffer</li><li>absolute EtOH</li><li>7...)
Current revision (02:47, 20 November 2009) (view source)
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>5 ml sterile LB medium (+ antibiotic)</li><li>P1 Buffer</li><li>P2 Buffer</li><li>ice-cold P3 Buffer</li><li>absolute EtOH</li><li>70% ethanol</li><li>TE</li><li>ddH<sub>2</sub>O</li><li>single bacterial colony</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Electrophoretic unit</li><li>Flasks of appropriate volumes</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>DAY 1</font></b><br>Inoculate <font color=#357EC7>5 ml sterile LB medium (+ antibiotic)</font> with <font color=#357EC7>single bacterial 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><b><font size=3>DAY 2</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>culture</font> into Eppendorf tube (1).<br>Centrifuge at a speed of <font color=#357EC7>8000 rpm</font> for <b><font color=#357EC7>2 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>100 µl</font></b> of <font color=#357EC7>P1 Buffer</font>.<br>Resuspend P1 Buffer by vortexing/by shaking vigorously.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>P2 Buffer</font>.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for at most <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>ice-cold P3 Buffer</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>5 - 10 mins</font></b>.<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>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>Add  <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>absolute EtOH</font> to Eppendorf tube (2).<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 - 20 mins</font></b>.<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>4°C</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% 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></ol></li></p><p><li><p><b>Option 1: </b>Dry the pellet in air. <br>(or)<br><b>Option 2: </b>Dry the pellet in speedvac. <br></p><p><font color = "#800517"><i>Do not overdo drying, or the pellet will not properly dissolve again.</i></font><br></li></p><p><li><p><b>Option 1: </b>Add <b><font color=#357EC7>30 µl</font></b> of <font color=#357EC7>TE</font>.<br>(or)<br><b>Option 2: </b>Add <b><font color=#357EC7>30 µl</font></b> of <font color=#357EC7>ddH<sub>2</sub>O</font>.<br></p><p>Resuspend ddH<sub>2</sub>O by vortexing/by shaking vigorously.<br>Perform agarose gel electrophoresis of appropriate quantity of  ddH<sub>2</sub>O mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.<br></li></p></ol></html>
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>sterile LB medium (+ antibiotic)</li><li>P1 Buffer</li><li>P2 Buffer</li><li>ice-cold P3 Buffer</li><li>absolute EtOH</li><li>70% ethanol</li><li>TE</li><li>ddH<sub>2</sub>O</li><li>single bacterial colony</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Electrophoretic unit</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>DAY 1</font></b><br>Inoculate <font color=#357EC7>5 ml sterile LB medium (+ antibiotic)</font> with <font color=#357EC7>single bacterial 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><b><font size=3>DAY 2</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>culture</font> into Eppendorf tube (1).<br>Centrifuge at a speed of <font color=#357EC7>8000 rpm</font> for <b><font color=#357EC7>2 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>100 µl</font></b> of <font color=#357EC7>P1 Buffer</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>5 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>P2 Buffer</font>.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for at most <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>ice-cold P3 Buffer</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>5 - 10 mins</font></b>.<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>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>Add  <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>absolute EtOH</font> to Eppendorf tube (2).<br>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>10 - 20 mins</font></b>.<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>4°C</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% 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></ol></li></p><p><li><p><b>Option 1: </b>Dry the pellet in air. <br>(or)<br><b>Option 2: </b>Dry the pellet in speedvac. <br></p><p><font color = "#800517"><i>Do not overdo drying, or the pellet will not properly dissolve again.</i></font><br></li></p><p><li><p><b>Option 1: </b>Add <b><font color=#357EC7>30 µl</font></b> of <font color=#357EC7>TE</font>.<br>(or)<br><b>Option 2: </b>Add <b><font color=#357EC7>30 µl</font></b> of <font color=#357EC7>ddH<sub>2</sub>O</font>.<br></p><p>Resuspend pellet by vortexing/by shaking vigorously.<br>Perform agarose gel electrophoresis of appropriate quantity of  ddH<sub>2</sub>O mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 13 hrs, 7 mins</font></b></p></html>

Current revision

Solutions/reagents:

  • sterile LB medium (+ antibiotic)
  • P1 Buffer
  • P2 Buffer
  • ice-cold P3 Buffer
  • absolute EtOH
  • 70% ethanol
  • TE
  • ddH2O
  • single bacterial colony

Equipment:

  • Incubator
  • Centrifuge
  • Electrophoretic unit
  • Eppendorf tubes

Steps:

  1. DAY 1
    Inoculate 5 ml sterile LB medium (+ antibiotic) with single bacterial colony and incubate with shaking for 12 hrs(overnight) at 37°C.
  2. DAY 2

    1. Measure out 1.5 ml of culture into Eppendorf tube (1).
      Centrifuge at a speed of 8000 rpm for 2 mins at room temperature, gently aspirate out the supernatant and discard it.
    2. Add 100 µl of P1 Buffer.
      Resuspend pellet by vortexing/by shaking vigorously.
      Store at room temperature for 5 mins.
    3. Add 200 µl of P2 Buffer.
      Store at room temperature for at most 5 mins.
    4. Add 150 µl of ice-cold P3 Buffer.
      Vortex the mixture for a few secs.
      Store the tube on ice for 5 - 10 mins.
    5. Centrifuge at maximum speed for 10 mins at 4°C and aspirate out the top layer.
      Transfer top aqueous layer into Eppendorf tube (2).
      Discard bottom layer.
    6. Add 2 volumes absolute EtOH to Eppendorf tube (2).
      Store the tube on ice for 10 - 20 mins.
    7. Centrifuge at maximum speed for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
    8. Add 1 ml of 70% ethanol.
      Mix solution by pipetting up and down several times.
    9. Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
  3. Option 1: Dry the pellet in air.
    (or)
    Option 2: Dry the pellet in speedvac.

    Do not overdo drying, or the pellet will not properly dissolve again.

  4. Option 1: Add 30 µl of TE.
    (or)
    Option 2: Add 30 µl of ddH2O.

    Resuspend pellet by vortexing/by shaking vigorously.
    Perform agarose gel electrophoresis of appropriate quantity of ddH2O mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 7 mins

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