Harmer Lab:Protocols:Y2H

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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

  1. Resuspend 1 colony AH109 in 50μL sterile dH2O
  2. Add 273μL PEG/LiAc mix, 5μL prey miniprep, 5μL bait
  3. Heatshock 15min, 45°C
  4. 8000rpm, 1min
  5. Take off Supernatant
  6. Add 1 ml YPDA + leave O/N
  7. 8000rpm, 1min
  8. Take off Supernatant
  9. Resuspend in 200μL 0.9% NaCl (filter sterilised)
  10. 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

0.2% Adehemisulphate

20mg in 10ml

PEG/LiAc Mix

800μL 40% PEG 100μL LiAc 100μL TE

Handy Hints

Rinse DO supplement off weigh boat into media

Dissolve w/stirring for 15min, then autoclave 121°C, 15min