Harmer Lab:Protocols:Y2H: Difference between revisions
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<h3><font style="color:red'">Yeast 2-Hybrid Transformation</font></h3> | <h3><font style="color:red'">Yeast 2-Hybrid Transformation</font></h3> | ||
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#Resuspend 1 colony AH109 in | #Resuspend 1 colony AH109 in 50μL sterile dH<sub>2</sub>O | ||
#Add 273μL PEG/LiAc mix, 5μL prey miniprep, 5μL bait | |||
50μL sterile dH<sub>2</sub>O | |||
273μL PEG/LiAc mix | |||
5μL prey | |||
5μL bait | |||
#Heatshock 15min, 45°C | #Heatshock 15min, 45°C | ||
#8000rpm, 1min | #8000rpm, 1min | ||
#Take off Supernatant | #Take off Supernatant | ||
#Add 1 ml YPDA + leave O/N | #Add 1 ml YPDA + leave O/N | ||
#8000rpm, 1min | #8000rpm, 1min | ||
#Take off Supernatant | #Take off Supernatant | ||
#Resuspend in 200μL 0.9% NaCl (filter sterilised) | #Resuspend in 200μL 0.9% NaCl (filter sterilised) | ||
#Plate 100μL onto Selective and Non-selective plates | #Plate 100μL onto Selective and Non-selective plates | ||
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Revision as of 12:42, 30 March 2009
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Room 2123 |
Yeast 2-Hybrid Transformation
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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):
