Harmer Lab:Protocols:Y2H: Difference between revisions
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50μL sterile dH<sub>2</sub>O | 50μL sterile dH<sub>2</sub>O | ||
273μL PEG/LiAc mix | 273μL PEG/LiAc mix | ||
5μL prey | 5μL prey | ||
5μL bait | 5μL bait | ||
Revision as of 12:37, 30 March 2009
Room 2123 |
Yeast 2-Hybrid TransformationResuspend 1 colony AH109 in; 50μL sterile dH2O 273μL PEG/LiAc mix 5μL prey 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
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)46.7g Min. SD Agar base 0.64g -L/-T DO Supplement Selective Plates (1 litre)46.7g Min. SD Agar base 0.60g -H/-L/-T DO Supplement 1ml α-gal |
Handy HintsRinse 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):