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thebeez1 at my-deja.com thebeez1 at my-deja.com
Thu Jan 18 14:43:55 EST 2001

In article < at mailbox40.utcc.utoronto.ca>,
  debbie.field at utoronto.ca (Deborah Field) wrote:
> Hi Folks,
> 	  I am working on an essential multi-domain gene. Either of two exclusive
> domains are sufficient to maintain vegetative growth.  I have made a URA3
> disruption (it's viable) and wish to replace MFG::URA with tagged alleles
> in the chromosome. Has anyone had success with a one-step transformation
> onto non-selective media followed by replica plating onto FOA? Because of
> the multi-domain complementation, I don't want any partial duplications.
> Relocation of the tagged genes to another locus is an alternative, I
> admit,  but would require more work.
> Thanks.
> Deborah Field

            Although the alleles were different, I did something simmilar
to this in that I had a gene that was disrupted with HIS3 that resulted
in a demonstrable decrease in growth rate.  I did a one step
transformation and isolated the colonies that grew "fast" over the
background then scored for his auxotrophs.  It worked, if I remeber right
of the 8 clones that I screened for two different tagged alleles
all 8 were confirmed by southern analyses.  I would imagine that
using 5-FOA would preclude having to score, so it might be even easier.

									Alexander Beeser


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