5 Ways to Destroy Your Yeast Transformation
Transforming yeast with DNA is a very similar process to transforming E. coli, but with just enough differences to trip you up if you let your attention slip. Whether you’re doing a yeast two-hybrid screen, or using yeast as a model system, here are a some mistakes to to avoid…
1. Forgetting to add single stranded DNA
While E. coli readily takes up double-stranded DNA, yeast requires the addition of single-stranded “carrier DNA” to enhance uptake of your plasmid or fragment. If you only add your plasmid to the transformation mix, chances are you’ll be confronted with a pristinely sterile plate after three days of incubation.
2. Using old PEG
PEG (polyethylene glycol) is a crucial ingredient in the yeast transformation buffer. Unfortunately, it’s annoying to make and goes bad quickly. The percentage of PEG will make or break the success of your transformation; an old PEG solution has had a chance to evaporate, so that the water to PEG ratio is off. If you can stand it, make new PEG for every transformation. Otherwise, make it in small batches, and seal tightly with parafilm between uses to minimize evaporation.
3. Using cells in stationary phase
Cells in log phase, or exponential growth, take up DNA with much better efficiency. This is not to say that cells from a stationary culture can’t be transformed – only that your successful transformation rate will be much lower. Your best bet is to use cells that are growing rapidly at the time of transformation.
4. Using the wrong selective marker
A stupid mistake, but one that I’ve made more times than I’d like to admit. Double check to save yourself some tears!
5. Cutting corners when heat-shocking
This is a major difference between E. coli and yeast transformations: while E. coli is relatively delicate, and requires a heat-shock of less than a minute to take up DNA, the cell wall makes yeast more hardy and resistant to shock. Thus, for efficient transformation, yeast are generally heat-shocked for up to 45 minutes. I’ve gotten away with as little as 15 minutes, but any shorter and the transformation efficiency is drastically reduced. If you’re not in a hurry, let it go the whole 45 minutes, or you’ll get to experience the joy of doing it all over again.
Do you have any tips for a successful yeast transformation?
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Hi
We’re looking into doing yeast transformation; do you happen to have a library of what failed yeast transformation plates look like vs. successful ones?
hello sir, i have one question here. if i add double stranded DNA as a carrier will the transformation occur? or what can i do? i have double stranded calf thymus DNA, can i boil and use it ? will that work? Please reply sir thank you……….
I worked in a big yeast genetics lab for 6 years and I have never heard of or observed PEG going bad. For our transformation buffer, we make 10X LTE and 44% PEG stock solutions. The dilute 10X LTE in 44% PEG 1:10, so final concentration is 1xLTE and 40% PEG. The 1X solution is good for up to 6 months and the stocks you can keep indefinitely. Using yeast in log phase is only important if you need really high transformation efficiency (2 hybrid or library screening). For routine transformations we dilute yeast 1:5 and grow 2-3 hours before transforming (not quite log phase). Also, adding DMSO to about 10% in your transformation mix can improve efficiency about 5 fold. When doing transformations, we incubate the transformation mix for 15 minutes with gentle mixing at 30 degrees, before a 15 minute heat shock.