Hi,
Im half way through performing a LexA based 2 hybrid screen. Ive pick
50 LEU+ and LAC+ positive clones. However, when I pick these from a
screen plate (GAL/RAF Xgal -His/ -Trp/ - Ura/ -Leu) and plate them on
glucose base drop out media (no library expression from the GAL1 drived
library plasmid) the colonies still go a pale blue - much paler than on
induction (galactose media) but still blue! The host strain with the
bait only (no libray/AD plasmid) doesn't activate any of the reporters
(LAZ or LEU). Am I seeing this pale blue because
i/ I recently pick the clones off an induction plate (galactose) and the
yeast still have residual library protein expression or Bgal expression.
ii/ The large streak of rapidly growing yeast have depleted the glucose
in the media around them and so the GAL1 promoter driving expression of
the libray/AD gene is no longer glucose repressed.
ii/ Some other reason?
If you have any ideas, Id be grateful of replies to my e-mail at
Mark.Bond at bris.ac.uk. I'll try to keep up to date with the news group
as well
Cheers
Mark Bond
PS Why not take a look at my web site. It has lots of useful molecular
biology protocols, links and my thesis plus lots of other fun stuff.
Its at
http://www.rajdoot.freeserve.co.uk/
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