Blackburn:Yeast Colony PCR v2.0 protocol: Difference between revisions
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New page: <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 nam... |
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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 | <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> </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> | ||
Revision as of 06:55, 20 November 2009
<html>
Solutions/reagents:
- 0.02M NaOH
- <a name="Q-solution">Q-solution
<tab>(5X)</a> - <a name="PCR buffer">PCR buffer
<tab>(10X)</a> - <a name="dNTPs">dNTPs
<tab>(10mM each)</a> - <a name="Forward primer">Forward primer
<tab>(100µM)</a> - Reverse primer
- Taq
- ddH2O
- a small colony
Parameters:
- X - 1 min/kbp of template (elongation time)
Equipment:
- Thermocycler
- Sterile 0.6-ml tubes
Steps:
- Yeast Cell Lysis
- Measure out 10 µl of 0.02M NaOH into sterile 0.6-ml microcentrifuge tube (1).
- 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. - Set the thermocycler to run the following program:
- 99°C, 10 mins
The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.
- Measure out 10 µl of 0.02M NaOH into sterile 0.6-ml microcentrifuge tube (1).
- PCR
- Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (2):
<thead></thead><tbody></body>Q-solution PCR buffer dNTPs Forward primer Reverse primer Taq ddH2O Colony PCR 2 µl 1 µl 0.2 µl 0.2 µl 0.2 µl 0.1 µl 5.3 µl - 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. - Add 1 µl of boiled sample.
A multichannel pipette is helpful here. - Program a standard thermocycler to run the reaction using the following parameters:
Initial denaturation- Denature: 95°C, 5 mins
- No. of cycles: 30
- Denature: 95°C, 10 secs
- Anneal: 50°C, 10 secs
- Elongate: 72°C, X
Termination- Elongate: 72°C, 10 mins
- Hold: 4°C, until removed from machine
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ -26 mins
</html> - Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (2):
