Blackburn:Yeast Colony PCR v2.0 protocol - OpenWetWare
Set aside a fresh sterile 0.6-ml microcentrifuge tube (3). Call it Master Mix aliquot.
Blackburn:Yeast Colony PCR v2.0 protocol
- Sterile 0.6-ml tubes
- 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:
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.
- Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (2):
| ||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|
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:
Elongation time : 1 min/kbp.I generally do 30 sec elongation.
- No. of cycles: 30
- Denature: 95°C, 10 secs
- Anneal: 50°C, 10 secs
- Elongate: 72°C, 60 secs
I don't think this step is critical.
- Elongate: 72°C, 10 mins
- Hold: 4°C, until removed from machine
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 6 mins