growth

Ronan O'Kennedy Ronan.okennedy at ucg.ie
Thu Feb 16 17:06:28 EST 1995


>Hi,
>
>Has anyone ever had problems growing yeast (with 2 selectable plasmids, 2u
>ori) in minimal medium?  My yeast grows well on plates and really slow
>(double time approx. O/N) in liquid medium (growth stops after 2 days at
>approx. OD600=3D1).  Apart from the yeast getting dizzy in the shaker, ther=
e
>shouldn't be any difference btwn liquid cultures and growth on solid medium=
..
>My LIQUID Bgal assays are not working out...HELP!
>
>The shaker is at 30C and 225rpm.
>
>Thanks for being there,
>
>Kath


Oh I forgot about the Bgal assays. The one I use is from  Guarente L,
Ptashne M (1981). Fusion of Escherichia coli  LacZ to the cytocrome-c gene
of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 78 : 2199-2203. and
it works fine.

put up to 500ul of culture in epp
make up to 1 ml with Z-buffer
add 50ul chloroform
add 20ul 0.1% SDS
vortex 10secs at high speed
preincubate at 28=B0C 5 min
add 200ul of freshly prepared 4mg/ml ONPG solution (in z-buffer)
incubate at 28=B0C
stop reaction with 500ul of 1M Na2CO3
spin at 13K for 2 mins
transfer aquous layer to cuvette and read absorbance at 420nm

Remember that the addition of the stop reagent is critical to colour
development so that standard
curves constructed using o-nitrophenol must also have the same final conc
of Na2CO3

Regards
Ronan



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**
Ronan O'Kennedy
Growth and Fermenatation Technology Lab
Dept of Microbiology
University College Galway
Ireland
Phone ***-91-24411ext 2191
=46ax   ***-91-25700
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**





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