Najeeb Siddiqui
riggs at scar.utoronto.ca
writes:
> I am looking for a protocol to show cut phenotype in yeast cells. We
> have cells that are supposed to show the cut phenotype but so far I am
> not able to clearly detect the phenotype using the glutaraldehyde fixed,
> DAPI stained cells. Any help will be much appreciated.
This may depend upon what species you are examining, and you haven't
told us the species or how you are making the cells cut, but here goes....
in pombe, there is no "protocol" to speak of. The cut phenotype is usually
generated by a mutation (temperature sensitive) and becomes apparent
when the cells are shifted to the restrictive temperature. Not all
the nuclei will be bissected by the division septum (the classic
"cut" phenotype); however
abnormal mitosis and segregation will be apparent in most cells.
Cut phenotypes can also be generated by treating certain
checkpoint-defective mutants with hydroxyurea or DNA damaging agents,
or when germinating certain replication defective null mutants from spores.
Fixing the cells with ethanol is the easiest, but there should be
no problem seeing abnormal cells with glutaraldehyde or heat
fixation or even live stained with DAPI.
If you have a cut mutant strain and all the cells look normal (like wildtype)
at the restrictive temperature, then there is something wrong with your strain.
If you are looking at the permissive temperature, they should look okay.
I hope this helps.
--
-susan
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