Quick and easy bioengineering procedures: Yeast (S. cerevisiae) colony PCR
Hi all,
This is the last post in a series of 4 in which I want to share a quick and easy to use procedure for engineering a yeast strain. The idea is to go from the cloning stage, right after doing a PCR (or order) to get the DNA you want to clone, up to the process of transforming the yeast and selecting the positive clones. Keep in mind that the goal here is to make it quick and easy, so I'm targeting those who are not experimental biologists and want minimal lab work, or those who have a super wonderful, really cutting edge protocol, but maybe go overwhelmed in terms of time and resources and want to simplify the cloning and transformation stages of the project.
In this last post on the process of engineering a yeast strain, I share a simple procedure to perform PCR directly from yeast colonies on an agar plate, namely yeast colony PCR. It is a very common procedure performed on bacterial colonies, but with yeast it is a bit tricky. I have never tried it on any species other than S. cerevisieae, and it is probably complicated to do it on other species, as the cell wall can complicate things.
Colony PCR is intended to detect positive clones beyond phenotype, and is the step one would do after transforming the cells, as shown in the third post in this series. This procedure for yeast is a bit more subtle than for bacteria. Basically, the lysis is done with NaOH for 20 minutes at 90C (I assume this is to break down the yeast cell wall), and the maximum recommended length for the amplicon is around 1kb. I have never used the resulting amplicon from colony PCR for sequencing, in the cases I want to sequence, I perform genome extraction using a commercial kit, and then amplify the purified DNA with high-fidelity PCR.
Briefly, the process consists of three steps. First, the yeast cells are lysed by incubating the small part of the colony in NaOH at high temperature, then the PCR itself is performed, and finally the product is resolved on an agarose gel. Once you have found your positive clones, the usual step is to sequence the introduced DNA to check for mutations, and finally store your engineered strain in glycerol at -80ºC. Then you have your strain and can phenotypically characterise and use it!
The detailed protocol goes as follows:
Yeast Cell Lysis
1. Aliquot 20uL of 0.02M NaOH into PCR tubes.
2. Pick a small part of the colony and resuspend in the NaOH.
3. Boil the samples on a PCR machine by incubating the tubes at 99C for 20 minutes
PCR
4. Prepare the master mix solution containing on ice, here what I use per reaction:
- Primers (at 10uL): 2uL each (so in total 4uL)
- Water: 9 uL
- 10 PCR buffer: 2uL
- dNTPs (10mM): 2uL
- Taq: 1uL
Total 18uL
5. Mix 18uL of the master mix solution with 2uL of boiled samples with the yeast sample
6. Run the following PCR cycle:
- Cell breakage: 95C, 5 minutes
Repeat 30 times:
- Denaturation: 95C, 30 seconds
- Annealing: 50C, 30 seconds (the temperature here would depend on the primers)
- Extension: 72C, 1 minute (1 minute for 1 Kb, I don’t recommend to go for longer
amplicons)
- Final extension: 72C, 10 minutes
7. Load the PCR product in a 1% agarose gel
Then, select the positives, those with the electrophoresis band at the proper size, according to its migration respect to a ruler.
GOOD LUCK! And don’t hesitate to contact me if anything needed.
Regards :)
Our protocol used essentially the same approach, but boiling only for 10 min, and we were particular about letting the samples sit at room temp for a while post-boil for the particulate matter to settle out before drawing from the top of the liquid for PCR.
Also notable, I've used this for frozen samples as well with good success -- scraping from a frozen culture, boiling, then using (for example) to quickly check if it's carrying the right plasmid :)