S pombe transformation: problems.

S L Forsburg forsburg at nospamsalk.edu
Thu Feb 27 10:57:37 EST 1997


Thomas Petit wrote:
> 
> Dears pombe lovers,
> 
> I have two questions:
> 
> 1_I would like to know if someone has already transformed S. pombe using a
> carbon source different than glucose (fructose, glycerol, gluconate....?)

I havent, since pombe doesnt grow so well on other sources.
Maybe Charlie Hoffman has?


> 2_I transformed (LiAc method) a diploid strain of S. pombe with two
> different constructions (for integration, previously linearised) in two
> different experiments, always changing my solutions of transformation, and
> I obtained in the two cases few transformants and more than two third are
> unable to sporulate!
> (I select on ade- to be sure to work with the diploid strain). Any idea??

If you are integrating your DNA, you need a fairly substantial amount
of homology to have it occur with good efficiency.   Also linear
fragments can rearrange and be maintained as unstable episomes, at
low frequency.

Second, your parent diploid may have gone spo-.  pombe diploids do
not want to be diploid, they want to be haploid.  They are poised
ready to sporulation, so forcing them to stay diploid is a strong
selection for sporulation defective mutants.  For this reason, 
I make diploids fresh when I need them and I don't rely on the ones
in the freezer to be sporulation competent.  Check your diploid by
streaking it out, replica plating to malt extract, and staining the
colonies two days later with iodine vapor.  Make sure you pick a
colony to work with (off the original plate, since iodine kills
the ones on  ME) that is iodine+ and sporulation competent.


--susan

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S L Forsburg          forsburg at salk.edu
The Salk Institute    http://flosun.salk.edu/~forsburg
La Jolla, CA



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