yeast colony pcr

Yeast Colony PCR

This protocol is based on the protocol specified by Michael Lisby at University of Copenhagen here.

The advantage of this protocol is that it is fast and simple, the disadvantage is that it seems to be less efficient for PCR products over 2000 bp. This protocol works best with fresh yeast on plates.

1. Book the PCR machine.

2. Prepare a PCR mix for the number of clones you have plus two negative (empty strain and no cells) and one positive control (if possible) and one extra for safe measure. The total volume of the PCR mixture should be 20 µL. We disregard the volume of the yeast cells, since it should be very small in comparison.

    for example, if you have ten clones to test,
    we make a mix for 13 reactions if we have
    a positive control.
   
    2x PCR mastermix        10 * 13 = 130
    primer1 (10 µM)         2  * 13 = 26
    primer2 (10 µM)         2  * 13 = 26
    H2O                     6  * 13 = 78
   

   Leave the PCR mixture on ice.

3. Program the PCR machine. If you have the sequences of the primers and the template, use the WebPCR simulator and use the program that is given, but use 35 cycles instead of 30.

    For example:
   
    95°C	 10 min
   
    35 cycles of:
   
        95°C	 Denaturation 30 sec
        55°C	 Annealing    30 sec (adjust Annealing temperature and extension time according to results from webpcr)
        72°C	 Extension    30 sec
   
    Room temperature (The 4-12°C hold is noot necessary)
   

It is best to use hot-start PCR, so turn the machine on and pause in the initial denaturation step. Some inexpensive PCR tubes of lower quality has a tendency to pop open while adding them to a hot PCR block. In this case a normal start is preferred.

3. Using the tip of a yellow or white pipette tip, transfer a small amount of cells to the inside of a PCR tube.

4. Microwave for 2 min at max power.

5. Place tubes on ice to cool down.

6. Add 20 µL of the reaction mix to each tube. Be careful to switch pipette tip between each addition. The negative control should be the tube that you prepare last.

7. Store tubes at -20C or Run PCR directly .

Extra

This page on openwetware describe a number of variations on yeast colony PCR.

The mastermix should contain a thermostable polymerase with a compatible buffer, dNTPs and Mg2+ at two times the final desired concentration

On interesting variation is the The Blackburn: Yeast Colony PCR v2.0. This protocol use 20 mM NaOH and Qiagen Q-buffer which mainly contains Betaine (Trimethylglycine)

Betaine can be bought from Sigma 1.5 mL cost 28.50 euros and is 5M which is 5x.

This quantity is good for 1500 * 5 = 7500µL PCR mix or 7500/20 = 375.0 twenty µL PCR reactions. This means 28.50/375 = 0.076 or 7.6 cents per PCR reaction which might be ok.

This page has a list of PCR adjuvants with references.

Commonly used PCR adjuvants are: 5% Glycerol 5% DMSO 0.8 mg/mL BSA T4 Gene 32 Protein

Note

Colony PCR from yeast is a notoriously fickle protocol which can easily be seen by the many variations avaliable by searching the internet. It seems many users have to tweak the protocol for it to work. Test the protocol with a positive and negative control if you can. This protocol is probably one of the quickest, but works best with short products. If this does not work, consider protocols involving NaOH or enzymatic degradation of the cell wall. Some protocols are available here.b