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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 287colonies should be picked to find rare true-positives. As the frequency of truepositiveswill be unknown at this step, the goal will be to pick through all of thefalse-positives that are expected in the number of library transformants beingscreened. For example, if the number of library transformants was 10 6 , the goalwill be to pick through the number of false-positives expected in 10 6 diploids. Ifthe cDNA-independent false-positive frequency is one LYS + colony in 10 4 CFUplated, it will be necessary to pick at least 100 LYS + colonies to find a true-positivethat exists at a frequency of one in 10 6 .24. In general, test plates for auxotrophic reporter characterization lacking onlyleucine or lysine would automatically keep selective pressure for the presence ofthe prey and the corresponding bait plasmids. Using plates with fewer dropped-outcomponents would slightly accelerate the growth, and the potential loss of otherplasmids would not influence the results of the assay on these plates. However, atthe investigator’s discretion, Leu − and Lys − plates can be substituted for Ura − His −Trp − Leu − or Ura − His − Trp − Lys − .25. In some cases no positives are obtained from library screens. Reasons for thismight include inappropriate library source; an inadequate number of screenedcolonies (3–5,000,000 primary transformants.26. Transfer approximately the volume of one middle-sized yeast colony (2–3 µLpacked pellet); do not take more, or quality of isolated DNA will suffer. The masterplate does not need to be absolutely fresh: plates that have been stored for 5 dat 4°C have been successfully used.27. Modified versions of this protocol with extended elongation times were also foundto work; the variant given in this chapter has amplified fragments of as much as1.8 kb in pretty fair quantity.28. If the library being screened is based on pJG4-5 plasmid (and primers specific forthis plasmid are used in PCR mastermix), only clones containing Raf1 and Krit-1plasmids would produce products; for a pYesTrp2-based library, take RalGDSclone as positive control.29. Sometimes a single yeast cell will contain two or more different library plasmids. Ifthis happens, it will be immediately revealed by PCR. In this case two bands can beseparately isolated from the gel, and reamplified for subsequent characterization byretransformation against naïve bait. Also, after bacterial transformation an increasednumber of clones should be checked to avoid the loss of the “real” interactor.30. Note: only the forward primer, FP1, works well in sequencing of PCR fragments;the reverse primer will only work in sequencing from purified plasmid.In general, the TA-rich nature of the ADH terminator sequences downstream of