Yeast Growth and Maintenance

1. YPD medium. 20 g/L Difco peptone, 10 g/L yeast extract, 20 g/L agar (for plates only) Add H20 to 950 mL. Adjust pH to 5 8, autoclave, and cool to -55°. Add dextrose (glucose) to 2% (50 mL of a sterile, 40% stock solution per liter)

2. SD medium (see Note 3) 6.7 g/L Difco yeast nitrogen base without amino acids (Difco), 20 g/L agar (for solid medium only) Add H20 to 850 mL/L, add 100 mL of the appropriate 10X dropout solution, adjust to pH 5 8, autoclave, and cool to ~55°C Then add 50 mL of a sterile, 40% dextrose stock solution per liter of medium

1. Segregate multiple AD/library plasmids within a single colony.

2. Sort persistent positive clones into groups.

3. Choose one representative from each group.

4. Generate clones harboring only the AD/library plasmid.

• Restreak colonies

• PCR amplify inserts

• Restriction digest DNA

• Electrophorese samples

• Compare banding patterns

• Restreak colonies

5. Confirm two-hybrid interactions and identify false positives in transformation experiments.

■ Transform E coli ' Complement leu B ' Isolate plasmid

6. Discard false positives, save true positives.

7. Verify true positives with an independent method.

• Retransform plasmids

• Select for markers

• Assay for 3-gal activity

• Sequence cDNA inserts

• Express and coimmunoprecipitate protein

Fig. 4. Procedures for eliminating false positives

3. 10X Dropout solution- 10X dropout solutions contain all but one or more of the components in Table 2; which components are omitted depends on the selection medium desired. To prepare SD/-Trp/-Leu, for example, use a 10X dropout

Table 1

Plasmids in the Two-Hybrid System

Table 1

Vector

Description

Size, kb

Refs.

pGBT9

GAL4 (|_|47) DNA-BD,ra/5/, ampr

5.4

52

pGAD424

GAL4 (768-ssi) AD, LEU2 ampr

6.6

57

pVA3

GAL4 BD/mouse p53

6.4

10

pTDl

GAL4 AD /SV40 large T antigen

15

9

pLAM5'a

GAL4 BD/hmnan Lamin C

6.3

52

pCLl

wild-type, full-length GAL4

15

52

0pLAM5' was modified by Clontech from the original plasmid pLm5' by subcioning a human Lamin C fragment from a HIS-mAtked plasmid to pGBT9,

0pLAM5' was modified by Clontech from the original plasmid pLm5' by subcioning a human Lamin C fragment from a HIS-mAtked plasmid to pGBT9,

GAL4 b-d - GAL4 binding domain ? = primitif

I = fronstriplion lermirurlion signal A= GAL4 nuclear kxolizntiwi signal fioR V (tisu Xba I

Pvu II

Olli)

Fig. 5. Map of DNA-BD vector pGBT9,

fioR V (tisu Xba I

Pvu II

Olli)

GAL4 b-d - GAL4 binding domain ? = primitif

I = fronstriplion lermirurlion signal A= GAL4 nuclear kxolizntiwi signal

Fig. 5. Map of DNA-BD vector pGBT9, solution lacking Trp and Leu. 10X dropout solutions can be autoclaved and stored at 4°C for up to 1 yr,

4. \M3-Aminotriazole (3-AT) (Sigma) filter-sterilized.

5. 40% Dextrose, autoclaved or filter-sterilized (avoid prolonged or repeated autoclaving).

2.3. Yeast Transformation

1. 10 mg/mL Herring testes carrier DNA: sheared and denatured (Clontech).

2, 50% PEG 4000: (polyethylene glycol, average 3350 kDa mol wt; Sigma). Filter sterilize or autoclave. Avoid repeated autoclaving.

6AL4 orf ?

Fig. 6. Map of AD vector pGAD424.

3- 100% DMSO (Dimethyl sulfoxide; Sigma).

4. 10X TE buffer; 0.\M Tris-HCl, 10 mMEDTA, pH to 7.5, and autoclave.

5. 10X LiAc: lA/Lithium acetate (Sigma), pH to 7.5 with dilute acetic acid, and autoclave.

6. IX PEG/LiAc/TE solution: (polyethylene glycol/lithium acetate). Prepare fresh from stock solutions just prior to use. Use 50% PEG 4000: 10X LiAc: 10X TE; 8:1:1, respectively.

7. IX TE/LiAc solution: 40 % PEG 4000, IX TE, and IX LiAc. Prepare just before use from 10X stocks.

2.4. p-Galactosidase Assays

1. Z buffer: 6.1 g/L Na2HP04 • 7H20, 15.5 g/L NaH2PQ4 ■ H20, 0.75 g/L KC1, 0.246 g/L MgS04-7II20. Adjust pll to 7.0 and autoclave.

2. X-gal stock solution: Dissolve 5-bromo-4-chloro-3-indolyl-|3-D-galactopyrano-side (X-GAL) in N,N-dimethylformamide (DMF) at 20 mg/mL. Store in the dark at -20°C.

3. Z buffer/X-gal solution: Prepare fresh daily as needed. 100 mL Z buffer, 1.67 mL X-gal stock solution.

SnaB I

Aat II

Bgl I

= GAL4 acii¥Ql»n domain sequence = prom Die r

= transcrifHwn terminal ion sequence = SY40 large 1 antigen nuclei r localization signal ftoRV

SnaB I

Aat II

Sph I /find 111

Sta I

MSM)

Pvu I

Bgl I

= GAL4 acii¥Ql»n domain sequence = prom Die r

= transcrifHwn terminal ion sequence = SY40 large 1 antigen nuclei r localization signal ftoRV

Sma I (B4D Bamti I (S44) Sal IICS503 Pst I (8(0) Bgl II (8»)

Table 2

Component Stock Solutions for 10X Dropout Media

Table 2

Component Stock Solutions for 10X Dropout Media

Component

Stock concentration, mg/L

Sigma cat. no.

1 L-Isoleucine

300

1-7383

2 L-Valine

1500

V-0500

3 L-Adenine hemisulfate salt

200

A-9126

4 L-Arginine HCl

200

A-5131

5 L-Histidine HCl monohydrate

200

H-9511

6 L-Leucine

1000

L-1512

7 L-Lysine HCl

300

L-1262

8 L-Methionine

200

M-9625

9 L-Phenylalanine

500

P-5030

10 L-Threomne

2000

T-8625

11 L-Tryptophan

200

T-0254

12. L-Tyrosine

300

T-3754

13. L-Uracil

200

U-0750

4 Z buffer with (3-mercaptoethanol. Used only if the highest sensitivity level is desired. To 100 mL of Z buffer, add 0.27 mL of P-mercaptoethanol (Sigma)

5 Whatman #5 or VWR grade 413 paper filters. 75-mm diameter filters for use with 100-mm diameter Petri plates, 125-mm diameter filters for 150-mm plates (Or, special order 85-mm and 135-mm filters directly from Whatman )

6. Liquid nitrogen

7 O-nitrophenyl (3-D-galactopyranoside (ONPG) solution: Prepared fresh prior to use (Sigma) 4 mg/mL in Z buffer, mix well

8 lMNa2C03.

0 0

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