This protocol utilizes the Y190 yeast strain, that characteristically turns from white to red during growth due to ade- mutation. Baits were subcloned into the pAS1cyh2 vector, and Preys subcloned into pACT2 (5).
Method Article
Directed yeast two hybrid screening for MEKK1 interaction partners
https://doi.org/10.1038/nprot.2007.37
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This protocol utilizes the Y190 yeast strain, that characteristically turns from white to red during growth due to ade- mutation. Baits were subcloned into the pAS1cyh2 vector, and Preys subcloned into pACT2 (5).
Y190 yeast strain
YPAD medium
0.1M LiAc (Sigma)
ssDNA (2.0 mg/ml)
PEG 3350 (Sigma)
Sterile H2O
42 ˚C waterbath
SC minus plates (-LEU, -TRP)
SC minus plates (-LEU, -HIS, -TRP), supplemented with 50 mM 3AT and 20 μg/ml X-gal
Transformation
1 Inoculate 10 ml of YPAD medium with a single Y190 colony and incubate overnight at 30 ˚C. 55 ml of fresh YPAD are then cultured overnight to an OD600 of 0.3. Cells are then incubated by shaking at 200 RPM and 30 ˚C until they reach an OD600 of 0.7.
2 Harvest cells at 2000 RPM for 5 min at room temperature.
3 Aspirate the media, wash cells in sterile water, and spin at 2000 RPM at 25 ˚C for 5 min.
4 Aspirate water and resuspend cells in 1 ml of 0.1 M LiAc and transfer suspension to a fresh microfuge tube. Pellet cells at 6000 RPM for 15 s and aspirate the supernatant. Next, resuspend the pellet in 500 μl of 100 mM LiAc.
5 Boil a 1 ml sample of ssDNA (2.0 mg/ml) and chill on ice. Mix the yeast cell suspension and transfer 50 μl samples into fresh tubes for the interactions to be tested.
6 Pellet cells at 6000 RPM and aspirate supernatant.
7 Add the following gently over the cell pellet: 240 μl of PEG 3350 (50% w/v), 36 μl of 1.0 M LiAc, 25 μg of ssDNA (2.0 mg/ml), and 50 μl sterile water containing plasmid DNA for the interaction screen (0.1 – 2.0 μg). Vortex mixture until cell pellet is completely mixed into suspension.
8 Incubate the suspension at 30 ˚C for 30 min. Transform the yeast by heat shock at 42 ˚C for 15 min, mix suspension carefully and pellet cells at 6000 RPM for 15 s.
9 Finally, resuspend yeast cells in 200 μl of sterile water for plating.
Plating and selection
10 Plate yeast onto SC minus plates (-LEU, -TRP) and grow at 30 ˚C for 3 – 5 days.
11 Streak single colonies onto SC minus plates (-LEU, -HIS, -TRP) supplemented with 50 mM 3AT and 20 μg/ml X-gal.
12 Yeast are then grown for 3 – 5 days and scored for growth and color change.
5 d minimum
Supported by the National Institutes of Health (ES04151 and ES06376 to M.K.), the American Cancer Society (M.K.) and the Leukemia and Lymphoma Society of America (T.E.; SCOR grant 7332-06).
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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