Filename: :Y2HYBRID.doc Written by:R. Johnson (9/11/97)
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Protocol: Yeast-2-Hybrid Analysis For Protein-Protein Interactions
Prepare bait fusion construct to desired bait using standard molecular biological techniques
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Yeast Transformation
(Note: see “Quicky” E. Coli Transformation (COMCELL.DOC) for Alternative Method)
Transform pC97.Bait into HF7c yeast strain as described below.
Start a 5ml YPD culture with 100 µl of innoculum of yeast HF7c strain overnight at 30 oC with
shaking.
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Innoculate 100 µl of overnight culture into 50 ml YPD and incubate overnight at 30oC
shaking.
In AM incoculate 300 ml YPD culture with 50 ml of overnight culture. Incubate at 30 oC
with shaking for 3 hours
Pellet in GSA rotor at 5000 rpm (4000 g) 5 min
Resupsend pellet in 10 ml water, transfer to 50 ml conical tube.
Pellet for 5 min at 7000 rpm (full speed on benchtop)
Resuspend pellet in 1.5 ml (LiAc 100 mM//TE 7.5), store on ice
Prepare 200 µg of salmon sperm DNA in TE at 10 mg/ml by boiling for 20 min and add
to 1-5 µg DNA when cooled. use 2.0 ml tubes
Add 200 ul of yeast suspension to each tube.
Add 1.2 ml of 40% PEG, 1X TE, 100 mM LiAc fresh to each tube and incubate at 30 C
shaking for 30 min.
Add DMSO to 10 % and mix gently.
Heat shock for 15 min at 42 C
Chill on ice 5 min and centrifuge 5 sec to pellet cells ( can plate directly
Resuspend cells in 1 ml of TE and plate on CM -Leu plates; .1 ml of yeast/plate.
Incubate at 30 oC until colonies appear.
Controls (n=4)
Control 1
Plate out colonies on CM -LEU, -TRP plates (yeast should not grow).
Control 2
Plate out yeast containing bait vector on CM-Leu plates (Yeast should grow but
not turn blue in filter assays).
Control 3
Prepare yeast for gels to check for expression of gal4 fusion protein by Western
blot (see box below).
Control 4.
Gal yeast colony filter assay control (from Breeden, L. and Nasmyth, K. 1985
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Grow colonies containg bait vector on a 100 mm plate in CM-leu media
Use fos/jun as a positive control, and just pC97 in HF7c as a negative
control.
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Place filter paper (schleicher and schuell supported nitrocellulose) on plate
and allow paper to wet, especially around colonies.
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Lift off and submerge in liquid nitrogen for 5-10 seconds.
Eipper/Mains Protocol Manual
Filename: :Y2HYBRID.doc Written by:R. Johnson (9/11/97)
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Lay filter colony side up on whatmand 3 MM chromatography paper
soaked at .3 ml per square inch of 1 mg/ml X-gal in Z buffer( X-gal from a
100 mg/ml stock in DMF diluted in 60 mM Na2HPO4, 40 mM
NaH2H2PO4, 10 mM KCl, 1 mM MgSO4, and 40 mM -mercaptoethanol.
Incubate for 15 min to 2 days.
(Note: fos/jun should turn blue indicating + control. Yeast HF7c with pC97.bait colony on CMLeu, -His plates should not grow).
Preparation of Yeast for protein gel
(Clonetech protocol)
2 colonies were incoulated into 5 ml CM-Leu and grown over weekend at 30oC
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Measure OD units (.068 of 1:20) 7.5 OD total
Pour 5 ml culture into 5 ml of ice in a ss-34 tube pellet at 1000 g.
Resuspend in 5 ml ice cold water
pellet at 1000 g, freeze pellet on dry ice
Resuspend pellet in cracking buffer (8M urea,5%SDS,40mM Tris, pH6.8, .1 mM
EDTA, .4mg/ml bromophenol blue) at 60 oC containing -mercaptoethanol
(1µl/ml) X and PMSF (100 µl)
Transfer to a tube with 80 µl of glass beads, heat to 70 oC 10 min
Vortex for 1 min, spin at 14000 rpm for 5 min.
Keep tubes on ice
Pellets are boiled 5 min, vortexed 1 min, spun at 14,000 rpm and then sups are
combined.
Analyze by Western blot using antisera to Gal4 or to bait protein
Library Transformation
Start yeast HF7c with pC97.Bait colony in 5 ml of CM-Leu, for library transformation.
Grow overnight at 30 C.
Transfer 1 ml overnight culture into 200 ml CM-Leu and grow overnight >16 hours at 30
C.
Measure A600 culture and innoculate 500 ml prewarmed YPD (.2 OD/ml final) and grow
until OD is .5-.8) Typically 2.5 hours gives OD of 0.3, and 4.5 hours gives OD of 0.71.
Harvest cells at 5K for 5-10 min in a sterile tube in Sorvall centrifuge.
Wash cells with 100 ml of sterile water and combine into one bottle; spin at 5K for 5 min
Resuspend pellet in 50 ml LiSorb () and shake gently for 20 min at 30 C. (Don't go too
long)
Transfer to two 50 ml sarstedt tubes and spin at 3,000 rpm for 10 min.
Resuspend each pellet in .5 ml LiSorb and combine into one tube (3-4 ml total) keep on
ice.
Prepare carrier DNA by boiling 10 mg/ml salmon sperm DNA for 10 min, add 400 µl
DNA to 0.8 ml LiSorb, mix and cool to room temp. Add 40 µg DNA at room temp.
Add library to yeast suspension and incubate at 30 oC for 30 min with out shaking.
Eipper/Mains Protocol Manual
Filename: :Y2HYBRID.doc Written by:R. Johnson (9/11/97)
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Add 27 ml of 40% PEG3350 in 100 mM LiAc/TE pH8.0, mix by inversion and incubate
at 30 C for 30 min mixing every 5 min.
Heat shock at 42 C for 10 min.
Remove 1 and 5 µl alliquots and plate on CM-Leu,-TRP plates for transformation
efficiency; use these # to calculate total colonies screened. (A typical transformation
efficiency = ~5 x 104 to 105 colonies/µg DNA).
Add cell mixture to 250 ml SC-Leu-Trp-His and shake at 250 rpm at 30oC for 1-3 hours.
Pellet cells at 4K for 10 min. resuspend in 4 ml CM-Leu-Trp-His and plate out 3-400 µl
large plate. Grow for 3-5 days.
Colonies that grow after 3-5 days are tested for B-galactosidase activity.
Colonies are plated in duplicate on CM-Leu-Trp-His plates with Fos/jun as a positive
control for the filter assay. Grow at 30 oC for 2 days.
Filter assays of clones (same as for control above). See Y-Filter for filter assay protocol
Filter assays were done 2 X.
Streak out positive colonies on CM-Leu-Trp-His plate to get single colonies. Grow at 30
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C over weekend for transformation back into E.coli.
(Note: Fos-jun control should turn within 15 min. Clones should turned blue within 15 min to 1
day).
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Recovery of Plasmid From Yeast
Start 5.0 ml overnight culture at 30 oC from a single colony.
Spin down at 3000 rpm for 10 min and resuspend pellet in 1 ml SCE
Spin again
Resuspend in 200 µl SCE with 20 µl -mercaptoethanol and 8 µl zymolase 20T (500
µg/µl).
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Incubate 60 min at 37 oC, vortexing every 10-15 min.
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Add 400 l solution 2 mix by inversion and incubate on ice 5 min.
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Add 300 µl solution 3, vortex 10 sec, ice 5 min.
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Centrifuge 4 min in microcentrifuge.
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Transfer 800 µl supernatant to new 2.2 ml tube (avoid protein). Add equal volumes of
phenol:chloroform:IAA(12:12:1), vortex, repeat extraction until no protein is observed in
the interface.
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Transfer most of aqueous phase and add equal volume of ice cold isopropanol. vortex and
freeze at-20 oC for 30 min.
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Spin for 20 min and wash pellet with 70% Etanol.
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Dry pellet in speed vac for 15 min.
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Dissolve pellet in 100 µl TE buffer, pH7.5.
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Transform maximum efficiency DH5 cells with 1 or 5 µl and plate on LB-Amp plates.
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Pick colonies, start overnight cultures, prepare minipreps, and screen by digestion to
determine which colonies contain bait vector and which colonies contain desired positive
clones.
16. Sequence desired positive clones
Eipper/Mains Protocol Manual
Filename: :Y2HYBRID.doc Written by:R. Johnson (9/11/97)
Solutions:
(for media leave out agar)
SCE (500ml)
(1 M Sorbitol, 100 mM Na2Citrate, 60 mM Na2EDTA.2H20)
Sorbitol...................................91.1g
Na2Citrate...............................14.2 g
Na2EDTA.2H20 ......................11.16g
Water up to .............................500 ml
sterile filter
CM-Leu plates (1L)
YNB-AA/AS ..........................1.7 g
2NH4.SO4 ...............................5 g
water .......................................780 ml
bactoagar ................................20 g
NaOH (50 mg/ml) ..................2.0 ml (for plates only)
autoclave and add
20% glucose ...........................100 ml
AA dropout ............................100 ml
100X His ................................10 ml
100X Trp ................................10 ml
CM-Trp plates (1L)
YNB-AA/AS ..........................1.7 g
(NH4)2SO4 ..............................5 g
water .......................................780 ml
bactoagar ................................20 g
NaOH 50mg/ml ......................2.0 ml (for plates only)
autoclave and add
20% glucose ...........................100 ml
AA dropout ............................100 ml
100X His ................................10 ml
100X Leu................................10 ml
CM-Leu,Trp plates (1L)
YNB-AA/AS ..........................1.7 g
(NH4)2SO4 ..............................5 g
water .......................................790 ml
Bactoagar ...............................20 g
NaOH 50mg/ml ......................2.0 ml (for plates only)
autoclave and add
20% glucose ...........................100 ml
AA dropout ............................100 ml
100X His ................................10 ml
Eipper/Mains Protocol Manual
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Filename: :Y2HYBRID.doc Written by:R. Johnson (9/11/97)
CM-His,Leu,Trp plates (1L)
YNB-AA/AS ......................... 1.7 g
(NH4)2SO4 ............................. 5 g
water ..................................... 800 ml
bactoagar ............................... 20 g
NaOH 50mg/ml ..................... 2.0 ml (for plates only)
autoclave and add
20% glucose .......................... 100 ml
AA dropout ........................... 100 ml
YPAD (1L)
Yeast extract.......................... 10 g
Peptone.................................. 20 g
Adenine .................................
hemisulfate ............................ 30 mg
water up to............................. 900 ml
autoclave and add
20% Dextrose ........................ 100 ml
LiSORB
100 mM LiOAc, 10mM Tris pH 8.0, 1 mM EDTA, 1 M Sorbitol
solution 2 (10 ml)
(0.2 N NaOH, 1% SDS)
2 N NaOH ............................. 1 ml
10% SDS ............................... 1 ml
water ...................................... 8 ml
solution 3 (500 ml)
(3 M KOAc, glacial acetic acid)
5M KOAc.............................. 300 ml
glacial acetic acid .................. 57.5 ml
water up to............................. 500 ml
Eipper/Mains Protocol Manual
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Filename: :Y2HYBRID.doc Written by:R. Johnson (9/11/97)
Chemical
-mercaptoethanol
(NH4.)2SO4
Bactoagar
Dextrose
Glacial acetic acid
Glucose
Hemisulfate
His
KOAc
Leu
Na2Citrate
Na2EDTA.2H20
NaOH
Peptone
SDS
Sorbitol
Trp
YNB-AA/AS
Yeast extract
zymolase 20T
Eipper/Mains Protocol Manual
Vendor
Catalog #
ICN
32-092-1
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