Colin MacDiarmid writes:
>I need some help again from the newsgroup. I have mated two isogenic
>haploids and want to isolate a diploid, but obviously have no selection
>for this. I have read that mating tests distiguish a and alpha mating
>types in haploids by absence of growth i.e. halo on plates with the
>appropriate mating factor. It seems to me from reading this that it could
>be possible to select a diploid from a mating mixture by streaking out on
>a plate containing both mating factors, to which only a diploid would be
>insensitive. I realise this might not be a tight selection, but any
>significant difference in growth rate between haploid and diploid cells
>would be OK for me, since I'd do a sporulation test on probable diploid
>isolates.
>>Does it sound feasible, has anyone tried this? How much of the mating
>factors would I need to use? (I dont have access to a micromanipulator,
>so can't pick out zygotes, as someone suggested I do). Any help would be
>greatly appreciated.
>
We have found that all you need to do is to mix overnights of the a and
alpha parents and spot the mix down on a YEPD plate. After overnight
growth, half of the cells or more in the spot are diploid, so you can just
streak them out and test mating type (or sporulation) on a dozen or so
single colonies.
Michael Lichten
lichten at helix.nih.gov
