3
$\begingroup$

I would like to get myself up to speed with what is currently known to science about yeast transformation. Specifically, transformation of plasmids and linear DNA fragments. I am particularly interested in practical aspects, such as:

  • What is salmon sperm for? What if I don't use it? What if I use more?
  • What is the relationship between number of cells, amount of transforming DNA, and transformation efficiency?
  • How does choice of electroporation cuvette gap size affect efficiency?
  • What can I do to get more colonies after transforming my yeast?
  • What steps can I skip to make the transformation quicker if I am doing an easy transformation?
  • How do different genetic backgrounds and media affect efficiency? Are there any common ones that reduce efficiency substantially? Are there any uncommon ones that increase it substantially?
  • What is the effect of post-transformation "recovery" time (eg. culture 12 h without selection and then plate on selective medium)?
  • Is making spheroplasts worth it?
  • Does the same transformation protocol work for different species (S. cerevisiae, S. pombe, C. albicans, etc.)?
  • Is there a coherent theoretical framework for understanding what actually happens when yeast are transformed, as opposed to "you mix some chemicals with DNA and throw cells in, abra kadabra, transformants come out"? The sort of theory I'm asking about would be useful in predicting conditions for better efficiency.

I am not expecting you to comprehensively answer these questions in your answer. This would make for a very long answer, and each of these could be a question here in its own right.

Instead, I want the answer to name reference materials which can be studied to learn more about the points above. For example, recent reviews or textbooks by established experts in the fields would be quite useful.

$\endgroup$
  • $\begingroup$ If "what should I read to learn more about concept X" is not an appropriate question for this site, I would be happy to delete this and split it into many smaller questions. $\endgroup$ – Superbest Aug 27 '14 at 21:31
  • $\begingroup$ I think the question is absolutely ok. However, collecting a number of references takes time...I can look into this tomorrow when I have a little spare time. $\endgroup$ – Chris Aug 27 '14 at 22:16
4
$\begingroup$

I have never worked much with yeast, but I can still give some answers:

Salmon sperm is used as a the so called "carrier DNA". It is thought to bind to the yeast cell wall and thus prevents that the DNA which shall be transformed does so. This raises the transformation efficiency. See here for more details: "Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.".

Effect of the recovery time: In this time you typically give the cells the chance to recover from the relative rude treatment and to express the resistance gene (when used) before moving them to a selective medium. This raises the efficiency of the transformation.

Spheroblasts: As far as I remember they are terribly fragile and painful to handle. However, when you succeed making them, you have cells which take up high quantitaties of DNA which makes them pretty efficient.

I think these reviews are definitely worth looking at (they also contain a lot more references):

If you have a chance to get hands on a copy of "Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual", I think this will be very helpful.

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.