Sebastián Sosa Carrillo
posted about 2 months ago
I have developed my scientific career in different countries, going through several research groups and participating in projects with very different objectives, but my priority has always been, and still is, to have fun and enjoy what I do. In the technical sense, because of my interests, my studies and my research experience, I define myself as an interdisciplinary biologist who integrates experimental and computational approaches to decipher biological processes and develop biotechnological applications. From a less formal point of view, I would say that I think like a biologist who always keeps evolution in mind, and I have engineering tools to measure, design and build.

Quick and easy bioengineering procedures: Yeast (S. cerevisiae) transformation

Hi all,

This is the third 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 post I share a simple and efficient procedure to incorporate exogenous DNA into Saccharomyces cerevisiae cells, whether the goal is to integrate DNA into the chromosome or to introduce an episomal plasmid. In the context of this series of posts, this procedure would come after obtaining the DNA intended to be transformed into yeast from a miniprep of the E. coli cells generated in the second post. The procedure presented here is known as lithium acetate (LiAc)/single-stranded carrier DNA/polyethylene glycol (PEG) transformation. There are other ways to transform yeast, and in particular Saccharomyces cerevisiae, so depending on the type of yeast and the complexity of the transformed DNA, the most appropriate approach (spheroplast, electroporation, biolistics, ...) will have to be chosen. In my experience, I have used this method on strains derived from the widely used laboratory strain S288C (i.e. BY4742, 41 ...), to integrate linear DNA chunks up to 5Kbp, and plasmids up to 10Kbp. There are probably countless versions of the protocol presented below, this particular one was derived from Gietz, 2014.

The principle of this method is not fully demonstrated, but most models are based on the use of PEG together with lithium cations to attach the DNA to the cell wall and penetrate it, respectively, all assisted by heat shock. In addition, the presence of single-stranded carrier DNA, usually from salmon sperm (don't ask me why salmon, but better to leave it like this), turns out to be essential in my experience, and is most likely used to prevent foreign DNA from being degraded by nucleases inside the cell.

Some detail to keep in mind for the success of this method, and indeed whenever working with yeast, is that homologies of foreign DNA to the yeast genome will potentially lead to unwanted recombination, especially if the introduced DNA is linear. Everything can become a mesh if this is not taken into account. Therefore, try to avoid DNA sequence homologies unless you want to use them to integrate DNA into the chromosome. However, if that is the case, it is most efficient to transform the piece of DNA targeted for integration into the linear state, after restriction with appropriate enzymes in a separate reaction.

Once your DNA is ready to be transformed, whether it is a circular plasmid or a linear piece from a PCR or restriction reaction, the method is based on washing the cells from mid-exponential phase with water, then with LiAc, and mixing with PEG, single-stranded carrier DNA, and the desired DNA to transform. The mixture is then incubated at 30°C for 30 minutes, followed by another incubation for one to two hours at 42°C. After this, transformation should have occurred, and what follows is recovery in rich YPD medium at 30°C for two hours, and selection on plates. The procedure can take a day (excluding selection), and a couple of hours of dedicated work, but allows several reactions to be carried out in parallel, and without the need for competent cells.


The night before grow the yeast strains to transform in 5mL of YPD at 30 °C. From the overnight culture, dilute 100 times into 50mL of YPD fresh media and let it grow for 4 hours at 30 °C, shaking. (The OD600 should be between 0.3-0.4). Then follow this procedure:

  1. Harvest the cells by pelleting at 3000 rpm in a centrifuge for 10 minutes at room temperature and discard the supernatant (seems a lot? They always survive)
  2. In parallel, place a thawed tube of carrier DNA reagent in a boiling water bath for 5 minutes and chill immediately in ice.
  3. Resuspend the cells in 10 mL of pure water
  4. Harvest the cells by pelleting at 3000rpm in a centrifuge for 10 minutes at room temperature and discard the supernatant
  5. Resuspend the cells in 1 mL of 100mM LiAc and transfer to a 1.5 mL tube
  6. Pellet the cells at 3000 rpm for 10 minute, remove the supernatant, and resuspend the pellet in 400 µL of 100 mM LiAc
  7. Aliquot 50 µL of cell suspension into 1.5 mL tubes (One per transformation and controls)
  8. Pellet the cells at 3000 rpm for 1 minute and remove the supernatant
  9. Add to the pellet, IN THE ORDER GIVEN, 240 µL PEG 3350 (50% w/v), 36 µL LiAc (1.0M), 50µL single-stranded carrier DNA (2.0 mg/mL) and 34 µL of sterile water plus any plasmid DNA (up to 1 µg if it is episomal plasmid, and 1-4µg if it is linear DNA for integration), and mix the pellet by vortex mixing briskly until resuspended, no clumps (usually I transform directly the restriction product if it is the case)
  10. Incubate for 30 minutes at 30 °C
  11. Incubate the tube in a water bath at 42 °C for at least one or two hours. Temperature-sensitive strains can be left on the bench overnight, then carried on to the next step
  12. Centrifuge the transformation tube at 3000rpm for 1 minute at room temperature and remove the transformation mix with a pipette
  13. Resuspend by pipetting gently in 1 mL of YPD and incubate two hours at 30°C shaking.
  14. Harvest the cells by centrifuging at 3000 rpm 10 minutes
  15. Resuspend the cells in 200 µL of sterile water by pipetting gently
  16. Plate the cells suspension on selectable plates (20 µL in one plate, 180 µL in the other)

Then, select the positives and check them by colony PCR for yeast, that is a bit subtler than for bacteria. Basically, the lysis is done with NaOH during 20 minutes at 90C, and the maximal recommended length for the amplicon is of around 1kb. But I will explain you this in the next post.


Regards :)

REFERENCES: Gietz, R. D., & Woods, R. A. (2006). Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Yeast Protocol, 107-120.

Nick Gervaisabout 1 month ago

Hey Sebastián! Have you ever determined whether the extended heat shock (1 or 2h) affects overall efficiency? A while back I was trying to do this protocol in sort of a "bulk" setup, where I just increased all of the reagents to a certain factor so that I could essentially do like 50 transformations all in one tube (to increase the number of colonies), rather than aliquoting them out as you do in Step 7. However, when I would heat-shock the tube I think the heat wasn't getting distributed evenly in it (given it was like 40mL of volume), because the resulting efficiency was awful. Let me know your thoughts!

_._darachm_._ {LastName}about 1 month ago

One way to boost transformants is to recover without nitrogen in the media, but definitely glucose. I think it was like a 4x boost, and Mike Chambers also saw that it boosted transformants in his hands.

My understanding of the LiAcPEG transformation is that you're using the salt+PEG to precipitate the DNA onto the cells (and maybe get it in and amongst cell wall proteins), using the Li to up-regulate a lithium transporter, and using heat to put them in a "shields-up" growth arrest low-PKA physiology. Then you recover them in media that stimulates a dramatic endocytosis of the DNA. Fermentable carbon source is crucial, it probably contributes to a PKA spike. That probably stimulates a few pathways of transporter ubi and endocytosis to pull the DNA in. One of Gietz's masters students had done FISH of DNA after transformations to see it on the surface, then going inside the cell after recovery, but the image is ruined by the thesis publisher website.

Anyways, here's a review with way more about yeast transformation: The findings of Neukamm~2002 and Nevoigt~2000 are particularly relevant to this tweak.

There's probably room to improve on the technique. What is needed is (1) DNA precipitation on the surface and (2) endocytosis. If you play games with nutrients / "stresses" you can maybe boost the latter, and think it would be fascinating to play with the K28 killer toxin to see if you can use it to boost endocytosis even more......

_._darachm_._ {LastName}about 1 month ago

Nick! A fellow bench worker in the building swears by leaving them at 42C for a long time, past 1 hour, maybe 2 hours if they feel like it. They seem to think the more the better. But an old yeast geneticist also told me that it stimulates petites, but I don't know if that's true or superstition

Sebastián Sosa Carrilloabout 1 month ago

Hello Nick. Sorry, I didn't quantify efficiency for different heat shock durations. But definitely, homogenization of the mixture can be a problem. I remember well that if you don't vortex every time you add a reactant in step 9, you will end up with kind of bubbles of PEG. Thus, vortexing (mixing well...) every time you add a volume in step 9 is very recommended (crucial probably).

Sebastián Sosa Carrilloabout 1 month ago

BTW, does someone know why the salmon sperm? I won't ask for results with other sperm sources, lol

_._darachm_._ {LastName}about 1 month ago

Yep I tried being gentle during mix and it didn't help. Vortex! But a multi/mastermix works in my hands too.

A old postdoc told me salmon sperm is a cheap source of DNA. Maybe it's just an easy to harvest (from fish) liquid form of DNA? Is there another animal liquid that's got lots of DNA?

I found long unsheared ssDNA to be best! As goopy as possible. Could be to precipitate, or buffer the cations? But then why is long better?

Nick Gervaisabout 1 month ago

Awesome, lots of new ideas to try. The nitrogen dropout idea seems especially compelling. Thank you both!! Sebastián, is there any reason you add the reagents from the transformation mix in the particular order? I would normally prepare the master mix with everything in it first and then add it to the cell pellet at once.

Sebastián Sosa Carrilloabout 1 month ago

Hi Nick,

The order of the reagents is important to facilitate mixing (especially, in my experience, adding the PEG first). It is fully compatible with making a master mix, because the last thing to be added is the DNA, which is usually specific to each tube (transformation). So, in case you want to make a master mix, you would add everything except the DNA, which is the last thing in the suggested order, and is not usually included in the master mix. (I hope this is clear)

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