酵母转化问题参考

5 Ways to Destroy Your Yeast Transformation

by Emily Crow on 27th of July, 2011 in Cell / Tissue Culture

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 minut es, or you’ll get to experience the joy of doing it all over again.