-=> Quoting Heidi Moss to All <=-
HM> Using the Brent 2-Hybrid System I have so far successfully cloned
HM> by bait into the DNAbd plasmid, transfected EGY48 w/ pSH18-34, checked
HM> for self reactivity, done a western to detect presence of the fusion
HM> protein, proceeded to transfect lib.plasmid containing a Hela cell
HM> lib, made glycerol stocks and proceeded to screen for protein
HM> interaction by plating onto Gal/Raf -His, -Ura, -Trp, -Leu plates.
HM> For curiosity I also plated the same cells onto Gal/Raf -H,-U,-T and
HM> Glu -H,-U,-T. The yeast colonies that grew the next day were reddish
HM> in color, where as the yeast on the Glu -H, -U,-T were white and
HM> present in high density as expected. Most interesting was the Gal/Raf
HM> -H,-U,-T plate contained a lawn of white colonies as well as a smaller
HM> number of large reddish colonies. In all the plates the number of
HM> reddish colonies was the same so I assumed that these were in fact
HM> yeast that were growing on -Leu plates because of some relevant
HM> protein interaction. Subsequently I have tested for Gal dependence
HM> and no color change was observed. What is going on? Can anyone help
Are you using Saccharomyces? Red mutants can be
expressed with some adenine mutations such as ade2, these will
typically form normal size colonies. Also some heme/porphyrin
mutations can cause a red phenotype, but these would grow slow or
not at all without supplementation.
Could you explain what the Brent 2-hybrid system is for those
that are not familiar with what it is and what it is used for?
More details and particulars may help us solve the mystery....
/^\/^^^^\/^\ R. Todd Lorenz
| # @ | Ellicott City, MD
\ :: / T.LORENZ at GENIE.GEIS.COM
=\||/=
#@
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