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.
- Inoculate yeast in YEPD for making it competent. See procedure for making competent yeast here.
- 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.
- Count the number of colonies that grew on the 1:30,000 transformation control plates.
- Pick the positive clones to -Leu -Trp -His +3AT plates.
- 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.
- 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.
- Lyse clones on the YEPD plate for PCR and sequencing.
- Replica plate -Leu -Trp plate to YEPD with a filter. Put both plates into incubator.
- Record the phenotypes of the selective plates
- Perform X-Gal staining.
- Save -Leu -Trp plate for future.