PJ694A in a two hybrid screen

Tim Soellick soellick at mpiz-koeln.mpg.de
Tue Feb 29 06:16:28 EST 2000


Hi, Richard,

I personally have very good experiences with the PJ69-4A strain but never tried
the -ade selection. So my recommendation is that you should replica-plate your
positiv clones on omission medium lacking his, leu, trp + 5 mM 3-aminotriazol
and grow them again for 3 days and then test them for LacZ activity with an
filter test.
For the resulting clones there are several ways to analyse them

   * the hard core way: Rescue the plasmids from the every clone, transform them
     into E.coli and check 4-6 mini-Plasmids by restriction analyses (HinDIII
     works fine for most of pAD-vectors). If you find that all plasmids are
     identical according to the size of the insert do check the clones by
     retransforming them into PJ69-4A (i) together with your original bait
     construct (ii) together with empty vector (iii) together with p53 or SNF1
     (see below).
   * the expensive way: Use a suspension of your yeast clones in 10 mM NaOH and
     do a PCR reaction with specific primers for the bait (it should be there,
     otherwise forget the candidate!) and for the prey. Then you directly
     sequence the PCR and you might be able to see, which of the clones are
     identical. Use positiv/negativ controls in the PCR reaction as there might
     occure artefacts. Be carefully with primers i.e. in the GAL4 activation
     domain as this gene somehow still exist in PJ69-4A. So use primers which
     are only vector-derived!
   * another way (which I haven't tried yet, but I know that other did this):
     Rescue 10 - 20 of your candidates, test them, sequence them and use the
     sequence for Southern analysis to check, which of your clones are
     identical.

Good luck. For a positiv control we normally use pBD-SNF1 / pAD-SNF4 (Fields &
Song, 1989). If you need an aliquot of them please write me a message.

Richard Schneider wrote:

> Hi all.
>
> 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





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