Blackburn:Yeast Colony PCR v2.0 protocol

<html><h2>Solutions/reagents:</h2><ul type="circle"><li>0.02M NaOH</li><li> <a name="Q-solution">Q-solution <tab><div style="margin-right: 600px;">(5X)</div></a></li><li> <a name="PCR buffer">PCR buffer <tab><div style="margin-right: 600px;">(10X)</div></a></li><li> <a name="dNTPs">dNTPs <tab><div style="margin-right: 600px;">(10mM each)</div></a></li><li> <a name="Forward primer">Forward primer <tab><div style="margin-right: 600px;">(100µM)</div></a></li><li>Reverse primer</li><li>Taq</li><li>ddH2O</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><li>Yeast Cell Lysis <ol type="a"><li>Measure out 10 µl of 0.02M NaOH into a sterile 0.6-ml tube. </li><li>Add a small colony to 0.02M NaOH. Resuspend colony 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. </li><li>Set the thermocycler to run the following program: <ul><li>99°C, 10 mins</li></ul>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. </li></ol></li><li>PCR <ol type="a"><li>Use the following table as a checklist for preparing the reaction: <table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td> </td><td>Q-solution</td><td>PCR buffer</td><td>dNTPs</td><td>Forward primer</td><td>Reverse primer</td><td>Taq</td><td>ddH2O</td></tr></thead><tbody><tr><td>Colony PCR</td><td>2 µl</td><td>1 µl</td><td>0.2 µl</td><td>0.2 µl</td><td>0.2 µl</td><td>0.1 µl</td><td>5.3 µl</td></tr></body></table></li><li>Set aside a fresh a sterile 0.6-ml tube. Call it Master Mix aliquot. Measure out 9 µl of master mix solution into Master Mix aliquot. </li><li>Measure out 1 µl of boiled sample into Master Mix aliquot. A multichannel pipette is helpful here. </li><li>Program a standard thermocycler to run the reaction using the following parameters: Initial denaturation <ul><li>Denature: 95°C, 5 mins</li></ul>Thermocycling <ul><li>No. of cycles: 30</li><li>Denature: 95°C, 10 secs</li> <li> Anneal: 50°C, 10 secs</li> <li>Elongate: 72°C, X</li></ul>I generally do 30 sec elongation. Termination<ul><li>Elongate: 72°C, 10 mins</li><li>Hold: 4°C, until removed from machine </li></ul>I don't think this step is critical. </li></ol></html>

Blackburn:Yeast Colony PCR v2.0 protocol

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