Blackburn:Yeast Colony PCR v2.0 protocol

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Current revision (03:04, 20 November 2009) (view source)
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>0.02M NaOH</li><li> <a name="Q-solution">Q-solution <i><br><tab><div style="margin-right: 600px;">(5X)</div></i></a></li><li> <a name="PCR buffer">PCR buffer <i><br><tab><div style="margin-right: 600px;">(10X)</div></i></a></li><li> <a name="dNTPs">dNTPs <i><br><tab><div style="margin-right: 600px;">(10mM each)</div></i></a></li><li> <a name="Forward primer">Forward primer <i><br><tab><div style="margin-right: 600px;">(100µM)</div></i></a></li><li>Reverse primer</li><li>Taq</li><li>ddH<sub>2</sub>O</li><li>a small colony</li></ul><h2>Parameters:</h2><ul type="circle"><li>X - 1 min/kbp of template (elongation time)</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Sterile 0.6-ml tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Yeast Cell Lysis</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>0.02M NaOH</font> into sterile 0.6-ml microcentrifuge tube (1).<br></li></p><p><li>Add <font color=#357EC7>a small colony</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>If the solution is cloudy, you've added enough cells.</i></font><br><font color = "#800517"><i>I have been told adding too much yeast can inhibit the reaction.</i></font><br></li></p><p><li>Set the thermocycler to run the following program:<br><ul><li><b><font color=#357EC7>99°C</font></b>, <b><font color=#357EC7>10 mins</font></b></li></ul><font color = "#800517"><i>In the mean time, prepare the master mix for the PCR reaction.</i></font><br><font color = "#800517"><i>The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.</i></font><br></li></p></ol></li></p><p><li><b><font size=3>PCR</font></b><br><ol type="a"><p><li>Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (2):<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>Q-solution</font></td><td><font color=#357EC7>PCR buffer</font></td><td><font color=#357EC7>dNTPs</font></td><td><font color=#357EC7>Forward primer</font></td><td><font color=#357EC7>Reverse primer</font></td><td><font color=#357EC7>Taq</font></td><td><font color=#357EC7>ddH<sub>2</sub>O</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Colony PCR</font></td><td><b><b><font color=#357EC7>2 µl</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><b><font color=#357EC7>0.1 µl</font></b></td><td><b><b><font color=#357EC7>5.3 µl</font></b></td></tr></body></table></li></p><p><li>Set aside a fresh sterile 0.6-ml microcentrifuge tube (3). Call it Master Mix aliquot. <br>Measure out <b><font color=#357EC7>9 µl</font></b> of <font color=#357EC7>master mix solution</font> into Master Mix aliquot.<br></li></p><p><li>Add <b><font color=#357EC7>1 µl</font></b> of <font color=#357EC7>boiled sample</font>.<br><font color = "#800517"><i>A multichannel pipette is helpful here.</i></font><br></li></p><p><li>Program a standard thermocycler to run the reaction using the following parameters:<br>Initial denaturation<br><ul><li>Denature: <b><font color=#357EC7>95°C</font></b>, <b><font color=#357EC7>5 mins</font></b></li></ul>Thermocycling<br><ul><li>No. of cycles: <b><font color=#357EC7>30</font></b></li><li>Denature: <b><font color=#357EC7>95°C</font></b>, <b><font color=#357EC7>10 secs</font></b></li> <li> Anneal: <b><font color=#357EC7>50°C</font></b>, <b><font color=#357EC7>10 secs</font></b></li> <li>Elongate: <b><font color=#357EC7>72°C</font></b>, <b><font color=#357EC7>X</font></b></li></ul><font color = "#800517"><i>I generally do 30 sec elongation.</i></font><br>Termination<ul><li>Elongate: <b><font color=#357EC7>72°C</font></b>, <b><font color=#357EC7>10 mins</font></b></li><li>Hold: <b><font color=#357EC7>4°C</font></b>, until removed from machine </li></ul><font color = "#800517"><i>I don't think this step is critical.</i></font><br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ -26 mins</font></b></p></html>
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>0.02M NaOH</li><li> <a name="Q-solution">Q-solution <i><br><tab><div style="margin-right: 600px;">(5X)</div></i></a></li><li> <a name="PCR buffer">PCR buffer <i><br><tab><div style="margin-right: 600px;">(10X)</div></i></a></li><li> <a name="dNTPs">dNTPs <i><br><tab><div style="margin-right: 600px;">(10mM each)</div></i></a></li><li> <a name="Forward primer">Forward primer <i><br><tab><div style="margin-right: 600px;">(100µM)</div></i></a></li><li>Reverse primer</li><li>Taq</li><li>ddH<sub>2</sub>O</li><li>a small colony</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Sterile 0.6-ml tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Yeast Cell Lysis</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>0.02M NaOH</font> into sterile 0.6-ml microcentrifuge tube (1).<br></li></p><p><li>Add <font color=#357EC7>a small colony</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>If the solution is cloudy, you've added enough cells.</i></font><br><font color = "#800517"><i>I have been told adding too much yeast can inhibit the reaction.</i></font><br></li></p><p><li>Set the thermocycler to run the following program:<br><ul><li><b><font color=#357EC7>99°C</font></b>, <b><font color=#357EC7>10 mins</font></b></li></ul><font color = "#800517"><i>In the mean time, prepare the master mix for the PCR reaction.</i></font><br><font color = "#800517"><i>The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.</i></font><br></li></p></ol></li></p><p><li><b><font size=3>PCR</font></b><br><ol type="a"><p><li>Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (2):<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>Q-solution</font></td><td><font color=#357EC7>PCR buffer</font></td><td><font color=#357EC7>dNTPs</font></td><td><font color=#357EC7>Forward primer</font></td><td><font color=#357EC7>Reverse primer</font></td><td><font color=#357EC7>Taq</font></td><td><font color=#357EC7>ddH<sub>2</sub>O</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Colony PCR</font></td><td><b><b><font color=#357EC7>2 µl</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><b><font color=#357EC7>0.1 µl</font></b></td><td><b><b><font color=#357EC7>5.3 µl</font></b></td></tr></body></table></li></p><p><li>Set aside a fresh sterile 0.6-ml microcentrifuge tube (3). Call it Master Mix aliquot. <br>Measure out <b><font color=#357EC7>9 µl</font></b> of <font color=#357EC7>master mix solution</font> into Master Mix aliquot.<br></li></p><p><li>Add <b><font color=#357EC7>1 µl</font></b> of <font color=#357EC7>boiled sample</font>.<br><font color = "#800517"><i>A multichannel pipette is helpful here.</i></font><br></li></p><p><li>Program a standard thermocycler to run the reaction using the following parameters:<br>Initial denaturation<br><ul><li>Denature: <b><font color=#357EC7>95°C</font></b>, <b><font color=#357EC7>5 mins</font></b></li></ul>Thermocycling<br><ul><li>No. of cycles: <b><font color=#357EC7>30</font></b></li><li>Denature: <b><font color=#357EC7>95°C</font></b>, <b><font color=#357EC7>10 secs</font></b></li> <li> Anneal: <b><font color=#357EC7>50°C</font></b>, <b><font color=#357EC7>10 secs</font></b></li> <li>Elongate: <b><font color=#357EC7>72°C</font></b>, <b><font color=#357EC7>60 secs</font></b></li></ul><font color = "#800517"><i>Elongation time : 1 min/kbp.I generally do 30 sec elongation.</i></font><br>Termination<ul><li>Elongate: <b><font color=#357EC7>72°C</font></b>, <b><font color=#357EC7>10 mins</font></b></li><li>Hold: <b><font color=#357EC7>4°C</font></b>, until removed from machine </li></ul><font color = "#800517"><i>I don't think this step is critical.</i></font><br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 1 hr, 6 mins</font></b></p></html>

Current revision

Solutions/reagents:

Equipment:

  • Thermocycler
  • Sterile 0.6-ml tubes

Steps:

  1. Yeast Cell Lysis

    1. Measure out 10 µl of 0.02M NaOH into sterile 0.6-ml microcentrifuge tube (1).
    2. Add a small colony.
      Resuspend pellet by vortexing/by shaking vigorously.
      If the solution is cloudy, you've added enough cells.
      I have been told adding too much yeast can inhibit the reaction.
    3. Set the thermocycler to run the following program:
      • 99°C, 10 mins
      In the mean time, prepare the master mix for the PCR reaction.
      The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.
  2. PCR

    1. Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (2):

       Q-solutionPCR bufferdNTPsForward primerReverse primerTaqddH2O
      Colony PCR2 µl1 µl0.2 µl0.2 µl0.2 µl0.1 µl5.3 µl
    2. Set aside a fresh sterile 0.6-ml microcentrifuge tube (3). Call it Master Mix aliquot.
      Measure out 9 µl of master mix solution into Master Mix aliquot.
    3. Add 1 µl of boiled sample.
      A multichannel pipette is helpful here.
    4. Program a standard thermocycler to run the reaction using the following parameters:
      Initial denaturation
      • Denature: 95°C, 5 mins
      Thermocycling
      • No. of cycles: 30
      • Denature: 95°C, 10 secs
      • Anneal: 50°C, 10 secs
      • Elongate: 72°C, 60 secs
      Elongation time : 1 min/kbp.I generally do 30 sec elongation.
      Termination
      • Elongate: 72°C, 10 mins
      • Hold: 4°C, until removed from machine
      I don't think this step is critical.

    TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 6 mins

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