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Yeast two-hybrid library screen by transformation

Adapted from: Walhout, M. & Vidal, M. (2001). Methods 24, 297-306.

Note: we perform all our Y2H screens by mating. This procedure is much more reproducible, and uses only a fraction of the amount of cDNA library stock.

This protocol assumes using yeast strain MaV203. If using a different strains, adapt 3-AT concentrations and/or phenotypic assays.

Day 1

  • Inoculate yeast in YEPD for making it competent. See procedure for making competent yeast here.

Day 2

  • Perform a large scale transformation using 30 µg of cDNA library. Plate the yeast onto 30 -Leu -Trp -His +20mM 3AT plates, and use a -Leu -Trp plate for the transformation efficiency control.

Day 4

  • Count the number of colonies that grew on the 1:30,000 transformation control plates.

Day 5-7

  • Pick the positive clones to -Leu -Trp -His +3AT plates.

Day 9

  • Pick a swatch from those clones that are still positive on -Leu -Trp -His +3AT, and dissolve them in water. Then spot them onto fresh -Leu -Trp -His +3AT plates. Use small spots, as they need to be replica plated several times.

Days 12 and 15

  • Replica plate to fresh -Leu -Trp -His +3AT plates. This part of the procedure reduces false positives due to multiple plasmids in the yeast cells. If you want, you can go straight from the day 9 step to the next step and skip this procedure.

Day 18

  • Pick a swatch from each clone and dissolve in water. Then spot them onto the following plates:
    • -Leu - Trp
    • YEPD for sequencing
    • -Leu -Trp +5FOA
    • -Leu -Trp -His +20mM 3AT (replica clean once)
    • -Leu -Trp -Ura
    • -Leu -His +20mM 3AT + Cycloheximide (not for cDNA library) (replica clean once)
    • Spot 5 controls at the bottom
  • Make a glycerol plate.

Day 19

  • Lyse clones on the YEPD plate for PCR and sequencing.

Day 20

  • Replica plate -Leu -Trp plate to YEPD with a filter. Put both plates into incubator.

Day 21

  • Record the phenotypes of the selective plates
  • Perform X-Gal staining.
  • Save -Leu -Trp plate for future.