Blackburn:Yeast Colony PCR v2.0 protocol: Difference between revisions

Revision as of 08:51, 1 October 2009

<html>

Yeast Cell Lysis
1. Measure out 10 µl of 0.02M NaOH into a sterile 0.6-ml tube.
2. 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.
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.

PCR
1. Use the following table as a checklist for preparing the reaction:
  
  <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
2. 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.
3. Measure out 1 µl of boiled sample into Master Mix aliquot.
  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, X
  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.
</html>

	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