Blackburn:Yeast Colony PCR v2.0 protocol

Solutions/reagents: 0.02M NaOH Q-solution  (5X)  PCR buffer  (10X)  dNTPs  (10mM each)  Forward primer  (100µM) </a></li>Reverse primer</li>Taq</li>ddH2O</li>a small colony</li></ul> Equipment: Thermocycler</li>Sterile 0.6-ml tubes</li></ul> Steps: <ol>  Yeast Cell Lysis  Measure out <font color=#357EC7>10 µl  of <font color=#357EC7>0.02M NaOH into sterile 0.6-ml microcentrifuge tube (1). </li> Add <font color=#357EC7>a small colony. Resuspend pellet by vortexing/by shaking vigorously. <font color = "#800517">If the solution is cloudy, you've added enough cells. <font color = "#800517">I have been told adding too much yeast can inhibit the reaction. </li> Set the thermocycler to run the following program: <ul><font color=#357EC7>99°C , <font color=#357EC7>10 mins </li></ul><font color = "#800517">In the mean time, prepare the master mix for the PCR reaction. <font color = "#800517">''The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.'' </li> </ol></li>  PCR  Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (2):  </li>  Set aside a fresh sterile 0.6-ml microcentrifuge tube (3). Call it Master Mix aliquot. Measure out <font color=#357EC7>9 µl  of <font color=#357EC7>master mix solution into Master Mix aliquot. </li> Add <font color=#357EC7>1 µl  of <font color=#357EC7>boiled sample. <font color = "#800517">A multichannel pipette is helpful here. </li> Program a standard thermocycler to run the reaction using the following parameters: Initial denaturation <ul><li>Denature: <font color=#357EC7>95°C , <font color=#357EC7>5 mins </li></ul>Thermocycling <ul><li>No. of cycles: <font color=#357EC7>30 </li><li>Denature: <font color=#357EC7>95°C , <font color=#357EC7>10 secs </li> <li> Anneal: <font color=#357EC7>50°C , <font color=#357EC7>10 secs </li> <li>Elongate: <font color=#357EC7>72°C , <font color=#357EC7>60 secs </li></ul><font color = "#800517">Elongation time : 1 min/kbp.I generally do 30 sec elongation. Termination<ul><li>Elongate: <font color=#357EC7>72°C , <font color=#357EC7>10 mins </li><li>Hold: <font color=#357EC7>4°C , until removed from machine </li></ul><font color = "#800517">I don't think this step is critical. </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 1 hr, 6 mins