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pombe electroporation problem

S L Forsburg forsburg at nospamsalk.edu
Wed Apr 23 02:22:06 EST 1997


Karen Yoas wrote:

> Most of the tetrad products have been transformed with the plasmid
> using the LiOAc procedure except for the products of a particular
> mating.  I have 7 mutants which were each mated back to a wild type
> strain.  Five sets of tetrad products from each mating have been
> analyzed except for one mutant.

....which I'll call M7 for simplicity....
 
> I have tried LiOAc transformation and the electroporation procedure
> with no success for 1/2 of the progeny.  The plasmid is essential in my
> system . Two of the tetrad products transformed with the plasmid were tested and
> found to be wild type of all five sets of tetrad products from the
> mating.  I am unable to transform the other two tetrad products with
> the plasmid to test them.

So in cross M7 x wt, only 2 of each tetrad are transformable and this 
corresponds with the wt allele:  ie, your mutant M7 is untransformable
out of this cross.  Is this correct?

Is the parent mutant M7 transformable?
Does your mutant have an easily scored phenotype?
What marker is on the plasmid?

Three suggestions:  
1) protoplast transformation with lipofectin.  It's a pain, but it can
be
very efficient.  

2) If you can transform the parents, you can look for plasmid recovery
after meiosis.  pombe plasmids are not very stable through meiosis.  
REP-based plasmids will come through in about 10% of the progeny. 
Therefore if you do this, random spores will be the way to go and
you will need to have an indpendent way of identifying the mutant
offspring.  

Both these suggestions require that your strain be able to 
maintain a plasmid once it is in there.  These suggestions assume 
that  the problem is establishment rather than maintenance but this
may not be the case. Strains defective in DNA replication or
 chromosome segregation or other processes may all have defects in 
plasmid maintenance.

3) Alternatively, you might integrate the plasmid into the
wt parent chromosome and track it as a separate marker through
meiosis.  There are useful integrating vectors with or without
promoters available.  If your strain can't maintain a plasmid
this is probably the best way to go.


-- susan

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S L Forsburg, PhD
Molecular Biology and Virology Lab          
The Salk Institute, La Jolla CA 

forsburg at salk.edu
http://flosun.salk.edu/~forsburg/lab.html



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