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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 267reporters (see Note 6). Assessment of transcriptional activation requires thetransfer of yeast from master plates to a variety of selective (dropout) plates. Asterile toothpick is usually used to move cells from individual patches on themaster plate to each of the selective media. In some cases (particularly ingenomic-scale applications) a large number of colonies expressing numerouscombinations of bait and prey need to be examined. In this case especially, it isuseful to use a transfer technique that is made for a high-throughput analysis,such as the one described herein.1. Add approx 50 µL of sterile water to the wells of 96-well microtiter plate with asyringe-based repeater or multichannel pipet (e.g., use wells A1–C6 for sixcolonies each of the three transformations described in Subheading 3.1.1., step 8).Position a micropipet tip insert grid on the microtiter plate, and attach it with tape:the holes in the insert grid should be placed exactly over the wells of the microtiterplate (this is important for stabilization of the tips in the plate, and will allowsimultaneous removal afterward, thereby speeding the replica process).2. Use sterile plastic micropipet tips (or toothpicks) to pick six yeast colonies (1–2-mmdiameter) from each of the transformation plates a–c (see Subheading 3.1.1., step 12).Place the tips in the wells leaving them in a near-vertical position supported by theinsert grid until all the colonies have been picked.3. Swirl the plate gently to mix the yeast into suspension and remove the insert grid,thereby removing all the tips at once.4. Use a replicator to plate (see Note 7) yeast suspensions to new plates. Each spokewill leave a drop approximately equal to a 3 µL volume. Use the following plates:a. One Glu/CM Ura − His − (producing a master plate).b. Two Gal-Raff/CM Ura − His − plates (for X-Gluc and X-Gal overlay assays, totest for GusA and LacZ reporter activity, respectively).c. One Gal-Raff/CM Ura − His − Lys − (for scoring activation of the LYS2 reporter).d. One Gal-Raff/CM Ura − His − Leu − (for scoring activation of the LEU2 reporter).e. One Gal-Raff/CM Ura − His − Lys − Leu − (optional).5. Grow the plates at 30°C. After 1–2 d, put the Glu/CM Ura − His − master plate at4°C, and assay the two Gal-Raff/CM Ura − His − plates for the activation of GusAand LacZ. Grow the remaining plates at 30°C until very strong growth is observedon the three LYS2 and LEU2 selection plates by the positive controls (for up to 4 d,but typically within 2 d).6. Activation of the GusA and LacZ reporters is assessed qualitatively, using theyeast grown for 18–36 h on the two Gal-Raff/CM Ura − His − plates. Use oneplate for overlay with XGal agarose, and the second for overlay with XGlucagarose, as follows.a. Slowly release approx 5 mL chloroform (CHCl 3) from a glass pipet held nearthe inside of the plate (or slowly pour from a small bottle). The objective is notto smear the colonies by too vigorous a release of CHCl 3. Do not cover platewith lid. Incubate colonies completely covered in CHCl 3for approx 5 min.Caution: CHCl 3is a toxic chemical. Take precautions to avoid inhalation andskin contact. Wear gloves. The procedure must be done in a chemical hood.