Harmer Lab:Protocols:Y2H

Room 2123 Department of Plant Biology 1002 Life Sciences, One Shields Ave. University of California Davis Davis, CA 95616 Contact: slharmer at ucdavis.edu

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Yeast 2-Hybrid Transformation Resuspend 1 colony AH109 in 50μL sterile dH2O Add 273μL PEG/LiAc mix, 5μL prey miniprep, 5μL bait Heatshock 15min, 45°C 8000rpm, 1min Take off Supernatant Add 1 ml YPDA + leave O/N 8000rpm, 1min Take off Supernatant Resuspend in 200μL 0.9% NaCl (filter sterilised) Plate 100μL onto Selective and Non-selective plates

YPD (Starter) plates (1 litre) 20g Peptone 10g Yeast extract 20g Agar 950ml dH2O pH 6.5 Autoclave, cool to 55°C, add 50ml 40% dextrose (filter-sterilised) YPDA (1 litre) 20g Peptone 10g Yeast extract Autoclave, then cool to 55°C and add; 15ml 0.2% Adehemisulphate (Sigma A9126) 50ml 40% Dextrose (filter sterilised) Non-Selective Plates (1 litre) 1.7g Nitrogen base 5g Ammonium Sulphate 8g Agar 0.64g -L/-T DO Supplement Autoclave, then add; 50ml 40% Dextrose Selective Plates (1 litre) 1.7g Nitrogen base 5g Ammonium Sulphate 8g Agar 0.60g -H/-L/-T DO Supplement Autoclave and cool to 55°C 50ml 40% Dextrose 1ml α-gal

Handy Hints Rinse DO supplement off weigh boat into media Dissolve w/stirring for 15min, then autoclave 121°C, 15min

• Matthew A. Jones 14:12, 30 March 2009 (EDT):