On 1 Mar 2000 20:33:15 -0000, David Keszenman-Pereyra
<D.K-Pereyra at sheffield.ac.uk> wrote:
>"does anyone know, when your are using PJ694A for a two hybrid, how can I
>determine true positiv in the best way further? I did two hybrid screen
>with a good transformation efficiency. After selection on -His omission
>plates I got 400 positives clones. I replica plate this to -Ade an I got
>several clones with different color and size. Some are red and others be
>white to brown. What is now the best way to determin ethe true positives.
>One more question, have anyone a very good positiv control plasmid but p53
>largeT? Thank for answering
>Best reguards
>Richard"
>>If the bait plasmid is URA3 and the genomic library is LEU2:
>>Select for transformants on -UL (-ura and -leucine)
>Replica plate on -ULA (-UL and -adenine) and/or -ULH-3AT (-UL and -histidine plus 3-
>amino 1,2,4 trizole)
If you've got a lot of candidates at this stage (and it sounds like you have),
you can discard any bait-independent positives by streaking clones on to FOA-Ade
media (as long as your bait is URA3, e.g. pGBDU-C1). Anything that grows on this
is activating GAL-ADE2in the absence of bait, so you can ditch these. You can
also get rid of any clones that are flaky and flocculent in liquid culture -
you'll get the feel for these once you've done a few screens! I find that these
stepes tend to reduce the number of clones to something vaguely sensible (e.g.
>200 to 40-50).
If anyone hasn't had Philip James update e-mail about PJ69-4A, I can post it
here, as it contained a few useful hints.
Barry
>Replica plate positive clones (grown on -ULA and -ULH+3AT) on -UL plus alpha GAL (5-
>bromo-4chloro-3-indolyl-alpha-D-galactopyranoside)(either spread 100 ul of 2 mg/ml
>alpha GAL over the entire surface of a -UL plate using a glass spreader or add 1 ml of
>20 mg/ml to a 1000 ml -UL agar medium at 55 C and pour plates).
>>Postive candidates are blue and grown on -ULA and -ULH+3AT
>>Replica plate on FOA-L (FOA plates minus leucine)
>Replica plate on -L
> -UL
> -LA
> -LH
>>Positive clones are white on -L and cannot grown on -UL, -LA and -LH
>>>>Good luck !!!!
>>David Keszenman-Pereyra
>University of Sheffield
>Department of Molecular Biology and Biotechnology, D38
>Firth Court, Western Bank, Sheffield S10 2TN
>UK
--
Barry Young
School of Biological Sciences
University of Manchester
Manchester
UK
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