Matchmaker Gold Yeast Two-Hybrid System User Manual.pdf

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Page 1 of 41

Takara Bio USA

Matchmaker® Gold

Yeast Two-Hybrid

System User Manual

Cat. Nos. 630466, 630489, 630498, 630499

(042424)

  Matchmaker Gold Yeast Two-Hybrid System User Manual 
(042424) 
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Takara Bio USA, Inc. 
Page 2 of 41 
 
 
Table of Contents 
I.  Introduction & Protocol Overview.................................................................................................................................. 4 
II.  List of Components ......................................................................................................................................................... 7 
III.  Additional Materials Required .................................................................................................................................... 8 
IV.  General Considerations Regarding Yeast Two-Hybrid Libraries ............................................................................... 9 
V.  List of Abbreviations .................................................................................................................................................... 10 
VI.  Control Experiments ................................................................................................................................................. 11 
A.  General Considerations ............................................................................................................................................. 11 
B.  Protocol: Control Mating Protocol ............................................................................................................................ 12 
VII.  Cloning and Testing Bait for Autoactivation and Toxicity ....................................................................................... 14 
A.  Generate a Bait Clone ............................................................................................................................................... 14 
B.  Detecting Bait Expression ......................................................................................................................................... 15 
C.  Protocol: Testing Your Bait for Autoactivation ........................................................................................................ 15 
D.  Protocol: Testing Your Bait for Toxicity .................................................................................................................. 16 
VIII.  Two-Hybrid Library Screening Using Yeast Mating ................................................................................................ 17 
IX.  Analysis of Results.................................................................................................................................................... 21 
X.  Confirmation of Positive Interactions & Rescue of the Prey Plasmid .......................................................................... 22 
A.  Protocol: Yeast Colony PCR Analysis to Eliminate Duplicate Clones .................................................................... 23 
B.  Protocol: Rescue and Isolation of Library Plasmid Responsible for Activation of Reporters .................................. 23 
C.  Protocol: Distinguishing Genuine Positive from False Positive Interactions ........................................................... 24 
D.  Sequence Analysis of a Genuine Positive ................................................................................................................. 26 
E.  Biochemical Methods to Confirm Positive Interactions ........................................................................................... 27 
F.  Downstream Analysis ............................................................................................................................................... 27 
XI.  Troubleshooting Guide ............................................................................................................................................. 28 
XII.  References ................................................................................................................................................................. 30 
Appendix A: Mate & Plate Library Construction ................................................................................................................. 32 
Appendix B: Library Titering ............................................................................................................................................... 35 
Appendix D: Yeast Growth Media & Supplements .............................................................................................................. 37 
 
   

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Table of Figures

Figure 1. The two-hybrid principle. ........................................................................................................................................ 4

Figure 2. Reporter gene constructs in Matchmaker yeast strains. ........................................................................................... 5

Figure 3. Two-hybrid screening using Mate & Plate Libraries............................................................................................... 6

Figure 4. Simple, one-step PCR cloning with In-Fusion HD Cloning Plus Kits. ................................................................. 14

Figure 5. An example of a typical yeast zygote. ................................................................................................................... 18

Figure 6. High stringency screening of potential interactors. ............................................................................................... 20

Figure 7. Strategies for analyzing and verifying putative positive interactions. ................................................................... 22

Figure 8. Illustration of the activation of reporter gene expression in genuine and false positives. ..................................... 24

Figure 9. Using cotransformation on selective media to verify protein interactions ............................................................ 25

Figure 10. Map of pGBKT7-53 DNA-BD control plasmid. ................................................................................................. 36

Figure 11. Map of pGADT7-T AD control plasmid. ............................................................................................................ 36

Table of Tables

Table I. Mating the Pretransformed Control Strains ............................................................................................................. 11

Table II. Yeast Host Strain Genotypes .................................................................................................................................. 32

Table III. Phenotype Testing on Various SD Media ............................................................................................................. 32

Table IV. Components of Yeast Media Set 2 & Yeast Media Set 2 Plus ............................................................................. 37

Table V. Individual Yeast Media Pouches for Matchmaker Gold Protocols ........................................................................ 38

Table VI. Additional Media & Media Supplements Required for a Two-Hybrid Screen .................................................... 38

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I. Introduction & Protocol Overview

Principle of the Two-Hybrid Assay

In a Matchmaker GAL4-based two-hybrid assay, a bait protein is expressed as a fusion to the Gal4 DNA-binding

domain (DNA-BD), while libraries of prey proteins are expressed as fusions to the Gal4 activation domain (AD;

Fields & Song, 1989; Chien et al. 1991). In the Matchmaker Gold Yeast Two-Hybrid System (Cat. No.

630489), when bait and library (prey) fusion proteins interact, the DNA-BD and AD are brought into proximity to

activate transcription of four independent reporter genes (AUR1-C, ADE2, HIS3, and MEL1) (Figure 1).

This technology can be used to:

• identify novel protein interactions

• confirm putative interactions

• define interacting domains

Figure 1. The two-hybrid principle. Two proteins are expressed separately, one (a bait protein) fused to the Gal4 DNA-

binding domain (BD) and the other (a prey protein) fused to the Gal4 transcriptional activation domain (AD). In yeast strain

Y2HGold, activation of the reporters (AUR1-C, ADE2, HIS3, and MEL1) only occurs in a cell that contains proteins which interact and

bind to the Gal4-responsive promoter.

The Bait

To make your GAL4 DNA-BD/bait construct, we recommend using pGBKT7, which is available separately (Cat.

No. 630443) or as a component of our Matchmaker Gold Two-Hybrid System (Cat. No. 630489). To investigate

ternary protein complexes, we suggest you use pBridge (Cat. No. 630404), a three-hybrid vector that contains two

MCS regions so that you can express a Gal4 DNA-BD fusion and a second protein of interest that may act as a

"bridge" between bait and prey.

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Four Reporter Genes to Detect Protein Interactions

There are four integrated reporter genes under the control of three distinct Gal4-responsive promoters (Figure 2)

in Takara Bio’s Y2HGold Yeast Strain, which are used to detect two-hybrid interactions.

AUR1-C. A dominant mutant version of the AUR1 gene that encodes the enzyme inositol phosphoryl ceramide

synthase. AUR1-C is expressed in Y2HGold Yeast Strain in response to protein-protein interactions that bring the

GAL4 transcriptional activation and DNA binding domains into close proximity. In Saccharomyces cerevisiae, its

expression confers strong resistance (AbA

) to the otherwise highly toxic drug Aureobasidin A (Cat Nos. 630466

& 630499). This drug reporter is preferable to nutritional reporters alone, due to lower background

activity. For example, the use of this reporter alone results in considerably less background than a histidine

reporter alone.

HIS3. Y2HGold is unable to synthesize histidine and is therefore unable to grow on media that lack this essential

amino acid. When bait and prey proteins interact, Gal4-responsive His3 expression permits the cell to

biosynthesize histidine and grow on –His minimal medium.

ADE2. Y2HGold is also unable to grow on minimal media that does not contain adenine. However, when two

proteins interact, Ade2 expression is activated, allowing these cells to grow on –Ade minimal medium.

MEL1. MEL-1 encodes a-galactosidase, an enzyme occurring naturally in many yeast strains. As a result of two-

hybrid interactions, a-galactosidase (MEL1) is expressed and secreted by the yeast cells. Yeast colonies that

express Mel1 turn blue in the presence of the chromogenic substrate X-alpha-Gal (Cat. Nos. 630462 & 630462).

NOTE: X-alpha-Gal is not X-Gal and is not a substrate for β-galactosidase.

Three Different Binding Sites

Three promoters controlling the four reporter genes AUR1-C, HIS3, ADE2, and MEL1 in Y2HGold are unrelated

except for the short protein binding sites in the UAS region that are specifically bound by the Gal4 DNA-BD.

Thus, library proteins that interact with unrelated sequences flanking or within the UAS (i.e., false positives) are

automatically screened out.

Figure 2. Reporter gene constructs in Matchmaker yeast strains. In Y2HGold, the HIS3, ADE2, and MEL1/AUR1-C reporter genes are under the

control of three completely heterologous Gal4-responsive promoter elements—G1, G2, and M1, respectively. The protein-binding sites within the

promoters are different, although each is related to the 17-mer consensus sequence recognized by Gal4 (Giniger et al. 1985; Giniger & Ptashne,

1988).

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Matchmaker Screening Protocol Overview

The entire Matchmaker screening process (Figure 3) consists of the following steps:

• Step 1. Perform control experiments

• Step 2. Clone and test bait for autoactivation and toxicity

• Step 3. Screen Mate & Plate library

• Step 4. Confirm and interpret results

Figure 3. Two-hybrid screening using Mate & Plate Libraries. Your bait protein is expressed as a fusion with the Gal4 DNA-BD in yeast strain

Y2HGold. The high-complexity library, which expresses fusions with the Gal4 AD, is provided in yeast strain Y187. When cultures of the two

transformed strains are mixed together overnight, they mate to create diploids. Diploid cells contain four reporter genes: HIS3, ADE2, MEL1, and

AUR1-C, that are activated in response to two-hybrid interactions.

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II. List of Components

Matchmaker Gold Yeast Two-Hybrid System (Cat. No. 630489)

Store all Matchmaker vectors at –20°C, all yeast (S. cerevisiae) strains at –70°C, and all Yeast Media Pouches at

room temperature.

For the Yeastmaker™ Yeast Transformation System 2, store carrier DNA and control plasmid at –20°C, and all

other components at room temperature.

Matchmaker Vectors

•

50 µl

pGBKT7 DNA-BD Cloning Vector (0.1 µg/µl)

•

50 µl

pGADT7 AD Cloning Vector (0.1 µg/µl)

•

50 µl

pGBKT7-53 Control Vector (0.1 µg/µl)

•

50 µl

pGADT7-T Control Vector (0.1 µg/µl)

•

50 µl

pGBKT7-Lam Control Vector (0.1 µg/µl)

Matchmaker Yeast Strains

•

0.5 ml

Y2HGold Yeast Strain

•

0.5 ml

Y187 Yeast Strain

Yeastmaker Yeast Transformation System 2 (also available separately as Cat No. 630439)

•

2 x 1 ml

Yeastmaker Carrier DNA, denatured (10 mg/ml)

•

20 µl

pGBT9 (0.1 µg/µl; positive control plasmid)

•

2 x 50 ml

50% PEG

•

50 ml

1 M LiAc (10X)

•

50 ml

10X TE Buffer

•

50 ml

YPD Plus Liquid Medium

Complimentary Yeast Media Pouches

•

1 x 0.5 L

YPDA

•

1 x 0.5 L

YPDA with Agar

•

1 x 0.5 L

SD–Leu with Agar

•

1 x 0.5 L

SD–Trp with Agar

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III. Additional Materials Required

The following reagents are required but not supplied.

A. Mate & Plate Libraries

(several catalog items; see www.takarabio.com for current list)

Store all Mate and Plate Libraries and Control vials at –70°C.

5 x 1.0 ml Library Aliquots (Universal Human and Mouse are also sold as 2 x 1 ml vials)

1 x 1.0 ml Mate & Plate Control

Alternatively, you can make your own Mate & Plate library using our Make Your Own “Mate & Plate”

Library System (Cat. No. 630490).

B. Accessory Kits

• Yeastmaker Yeast Transformation System 2 (supplied with your system—see Section II; also sold

separately as Cat. No. 630439)

• Easy Yeast Plasmid Isolation Kit (50 preps; Cat. No. 630467)

• Matchmaker Insert Check PCR Mix 2 (Cat. No. 630497; for characterizing the cDNA inserts of

positive clones from your library screening.)

C. Tools for Plating Yeast

Tools for plating yeast include a sterile glass rod—and a bent Pasteur pipette or 5-mm glass beads for

spreading cells on plates. (Use 5–7 beads per 100-mm plate, or 15–20 beads for a 150-cm plate).

D. Yeast Media

Table IV (in Appendix D) lists the components of the Yeast Media Set 2 (Cat. No. 630494) and the Yeast

Media Set 2 Plus (Cat. No. 630495). These media sets contain a complete assortment of mixes for

preparing eight specialized broth and agar media, designed for use with the Matchmaker Gold Yeast Two-

Hybrid System, in convenient, “ready-mixed” foil pouches. The Yeast Media Set 2 Plus also contains the

additional media supplements Aureobasidin A and X--Gal, which are required for the protocols

described in this user manual. Table V (in Appendix D) contains information for purchasing each of the

media mixes separately, in packs of 10 pouches, and Table VI (in Appendix D) contains preparation

instructions for all additional required media supplements and information for purchasing Aureobasidin A

and X--Gal separately.

Additionally, the following should be considered when culturing yeast for a two-hybrid screen:

• See Appendix D for working and stock concentrations of Aureobasidin A and X--Gal.

• SD medium (synthetically defined medium) is minimal media that is routinely used for culturing

S. cerevisiae. SD base supplies everything that a yeast cell needs to survive (including carbon

and nitrogen sources). Essential amino acids, which are added to SD base to create minimal

medium, are already included premixed in Takara Bio’s Yeast Media Pouches. The particular

minimal medium that is chosen will determine which plasmids and/or activated reporters are

selected for.

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• SD/–Leu/–Trp dropout supplement is used to select for the bait and prey plasmids. SD/–Leu/–

Trp dropout is so called because the medium includes every essential amino acid except for

leucine and tryptophan, which are omitted from the formulation (or “Dropped Out”). Cells

harboring Matchmaker bait and prey plasmids are able to grow because the plasmids encode

tryptophan and leucine biosynthesis genes, respectively, that are otherwise absent from the cell.

We often refer to SD/–Leu/–Trp as Double Dropout (DDO) in this user manual.

• SD/–Ade/–His/–Leu/–Trp dropout supplement is used to select for the bait and prey plasmids,

and in addition, for the activation of the Gal-responsive HIS3 and ADE2 genes as part of the

confirmation step of the two-hybrid assay. Colonies that grow on this Quadruple Dropout

(QDO) contain both bait and prey plasmids and also express proteins that interact with each other

to activate HIS3 and ADE2. This medium is used at the end of the two-hybrid screen to confirm

interactions.

IV. General Considerations Regarding Yeast Two-Hybrid Libraries

Use of Mate & Plate Libraries

Please note that the protocols described in this manual assume that you are using a Mate & Plate Library. These

libraries utilize the natural ability of haploid yeast strains such as Y187 and Y2HGold to mate with each other to

form a diploid cell, providing a very easy way to introduce an entire library (prey) to your bait.

• Takara Bio strongly recommends using Mate & Plate Libraries with the Matchmaker Gold System.

These libraries provide by far the simplest method for yeast two-hybrid screening because no library-scale

transformations or labor-intensive amplifications are needed. Thus, very little optimization and hands-on

time are required.

• Several Mate & Plate libraries are available for purchase from Takara Bio, supplied as 5 x 1 ml vials (and

also sold as 2 x 1 ml vials for Universal Human and Universal Mouse libraries). Alternatively, you can

easily “Make Your Own Mate & Plate Library” using Cat No. 630490, and store enough 1 ml vials for

more than 100 library screens.

• A single 1-ml Mate & Plate Library aliquot is sufficient for each complete library screening (>1 x 10

independent clones).

• See Appendix A for more details on prey vectors used to construct Mate & Plate Libraries. These libraries

are supplied in Saccharomyces cerevisiae strain Y187, in Freezing Medium. Depending on which library

you purchase, your library may be cloned into pGADT7-Rec2, pGADT7-RecAB, or pACT2. (See

Certificate of Analysis for details.). All are compatible with Matchmaker Gold.

• A Mate & Plate Control is supplied with all Mate & Plate libraries. This control is Y187 Yeast Strain

pretransformed with our pGADT7-T positive control plasmid, which expresses the Gal4 AD-SV40 large

T-antigen fusion protein. See Section VI for control experiments.

• Once a library aliquot has been thawed, do not refreeze it. With every freeze/thaw cycle, there is a ~10%

loss in viability, which can affect the quality of the library.

• The recommended Freezing Medium consists of YPDA broth + 25% glycerol (see Appendix D).

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V. List of Abbreviations

AD fusion library

[or AD library]

A cDNA library (such as a Mate & Plate Library) constructed in an activation domain

(AD) vector such that the proteins encoded by the inserts are fused to the 3’ end of the

Gal4 AD

AD/library plasmid

Plasmid encoding a fusion of the Gal4 activation domain and a library cDNA

AD/library protein

A protein fusion comprised of the Gal4 activation domain and a polypeptide encoded by a

library cDNA

AD vector

Plasmid encoding the yeast Gal4 activation domain

DNA-BD vector

Plasmid encoding the Gal4 DNA-binding domain

DNA-BD/bait

plasmid

Plasmid encoding a fusion of the Gal4 DNA binding domain and a bait cDNA

DNA-BD/bait

protein

[or “bait”]

A protein fusion comprised of the Gal4 DNA binding domain and a polypeptide encoded

by a bait cDNA

Yeast Phenotypes

AbA

The antibiotic Aureobasidin A, which is toxic to yeast at low concentrations (0.1–0.5

µg/ml). It acts by inhibiting a yeast enzyme, inositol phosphoryl ceramide synthase.

AbA

Resistance to the antibiotic Aureobasidin A, conveyed by expression of the AUR1-C gene

product

AUR1-C

A dominant mutant version of the AUR1 gene, which encodes the enzyme inositol

phosphoryl ceramide synthase. This gene is expressed in yeast strain Y2HGold, in

response to protein-protein interactions that bring the GAL4 activation and binding

domains in proximity, thus conferring resistance to the antibiotic Aureobasidin A.

Ade–, or His–, or

Leu–, or Trp–

Requires adenine (Ade), or histidine (His) or leucine (Leu), or tryptophan (Trp) in the

medium to grow; i.e., is auxotrophic for one (or more) of these specific nutrients

LacZ

Expresses the LacZ reporter gene; i.e., is positive for ß-galactosidase (beta-gal) activity

Mel1

Expresses the MEL1 reporter gene; i.e., is positive for α-galactosidase (alpha-gal) activity

Miscellaneous

Minimal, synthetically defined medium for yeast; is comprised of a nitrogen base, a

carbon source (glucose unless stated otherwise), and a DO supplement

Dropout (supplement or solution); a mixture of specific amino acids and nucleosides used

to supplement SD base to make SD medium; DO solutions are missing one or more of the

nutrients required by untransformed yeast to grow on SD medium

DDO

Double dropout medium: SD/–Leu/–Trp

DDO/X/A

Double dropout medium: SD/–Leu/–Trp supplemented with X-alpha-Gal and

Aureobasidin A

TDO

Triple dropout medium: SD/–His/–Leu/–Trp or SD/–Ade/–Leu/–Trp

QDO

Quadruple dropout medium: SD/–Ade/–His/–Leu/–Trp

QDO/X/A

Quadruple dropout medium: SD/–Ade/–His/–Leu/–Trp supplemented with X-alpha-Gal

and Aureobasidin A

YPD

A blend of yeast extract, peptone, and dextrose in optimal proportions for growth of most

strains of S. cerevisiae

YPDA

YPD medium supplemented with adenine hemisulfate (1X concentration = 120 µg/ml)

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VI. Control Experiments

PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING. Use this procedure to perform a control

mating before screening a two-hybrid library.

A. General Considerations

To familiarize yourself with the procedures and expected results of a two-hybrid assay, use these

procedures to perform a control mating before you begin screening the library. Select for diploids and for

two-hybrid protein-protein interactors as described below.

• pGBKT7-53 encodes the Gal4 DNA-BD fused with murine p53; pGADT7-T encodes the Gal4

AD fused with SV40 large T-antigen. Since p53 and large T-antigen are known to interact in a

yeast two-hybrid assay (Li & Fields, 1993; Iwabuchi et al. 1993), mating Y2HGold [pGBKT7-

53] with Y187 [pGADT7-T] will result in diploid cells containing both plasmids that can activate

all four reporters (Table I).

• A negative control should also be performed using pGBKT7-Lam (which encodes the Gal4 BD

fused with lamin) and pGADT7-T. Diploid yeast containing pGBKT7-Lam and pGADT7-T will

grow on SD/–Leu, SD/–Trp and SD/–Leu/–Trp (DDO) minimal media, but no colonies should

grow on DDO + AbA.

• Table I indicates the selection media required for strains containing a DNA-BD vector, AD

vector, or both, as well as the selection for diploids expressing interacting proteins.

Table I. Mating the Pretransformed Control Strains

Mating Strain

[plasmid]

Plate on SD Minimal Agar

Medium

Selects for

MEL1

Phenotype

Y2HGold [pGBKT7-

53]

Y187[pGADT7-T]

–Leu

pGADT7-T

White

–Trp

pGBKT7-53

White

–Leu/–Trp

(DDO)

Diploids containing pGBKT7-53

and pGADT7-T

White

–Leu/–Trp/X-alpha-Gal/AbA

(DDO/X/A)

Diploids that have also activated

Aureobasidin A resistance and

a-galactosidase through protein-

protein interactions

Blue

Controls for mating efficiency.

Selects for diploids expressing interacting proteins.

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B. Protocol: Control Mating Protocol

1. Materials:

• SD/–Trp with Agar (see Appendix D)

• SD/–Leu with Agar (see Appendix D)

• SD/–Trp/X-alpha-Gal agar plates (see Appendix D)

• SD/–Leu/–Trp agar plates (see Appendix D)

• SD/–Leu/–Trp/X-alpha-Gal /AbA agar plates (see Appendix D)

• 2 x YPDA Broth (see Appendix D)

• YPDA Broth + 25% glycerol (Freezing Medium; see Appendix D)

• Y2HGold Yeast Strain (Bait Strain)

• Y187 Yeast Strain (Prey Strain)

• pGBKT7-53 Positive Control Bait Plasmid

• pGBKT7-Lam Negative Control Bait Plasmid

• pGADT7-T Positive Control Prey Plasmid

NOTES:

• Use the Yeastmaker Yeast Transformation System 2 (supplied with this system) for all

transformations.

• X-alpha-Gal is not the same as X-Gal.

2. Use the Yeastmaker Yeast Transformation System 2 according to the small-scale protocol in the

accompanying user manual to perform the following three transformations.

Strain

Transformation Plasmid

Plating Medium

Y2HGold

pGBKT7-53

SD/–Trp with Agar

Y2HGold

pGBKT7-Lam

SD/–Trp with Agar

Y187

pGADT7-T

SD/–Leu with Agar

3. Grow at 30°C for 3 days.

NOTE: If you wish, you may stop the experiment at this step and resume work later. The plates

can be stored at 4ºC in subdued lighting for up to one month.

4. Pick one 2–3 mm colony of each type for use with the following small-scale mating procedure

(Steps 5–7).

• Positive Control Mating: Y2HGold [pGBKT7-53] and Y187 [pGADT7-T]

• Negative Control Mating: Y2HGold [pGBKT7-Lam] and Y187 [pGADT7-T]

5. Place both colonies in a single 1.5-ml centrifuge tube containing 500 µl of 2X YPDA and vortex

to mix.

6. Incubate with shaking at 200 rpm at 30°C overnight [20–24 hr].

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7. From the mated culture (0.5 ml), spread 100 µl of 1/10, 1/100, and 1/1,000 dilutions on each of

the following agar plates. Incubate plates (colony side facing downward) at 30°C for 3–5 days.

• SD/–Trp

• SD/–Leu

• SD/–Leu/–Trp (=DDO)

• SD/–Leu/–Trp/X-alpha-Gal/AbA (=DDO/X/A)

8. Expected results after 3–5 days:

Positive control:

• Similar number of colonies on DDO and DDO/X/A agar plates

• Colonies on DDO/X/A are blue

Negative control:

• Colonies on DDO, but no colonies on DDO/X/A agar plates

NOTES:

• For positive interactions, theoretically, the number of colonies should be the same on both

media. DDO selects for the presence of both plasmids (i.e., proper mated diploids) and

DDO/X/A selects for the plasmids as well as for the interactions of the hybrid proteins

encoded by them to activate the AbA

and MEL1 reporters. However, a difference

(approximately 10–20% lower on DDO/X/A) is usually observed.

• If you see no colonies on DDO, compare to colony counts on SD/–Trp and SD–/Leu single

dropout media to determine if there was a problem with the bait or the prey cultures,

respectively.

9. Pick healthy 2-mm colonies from DDO plates, restreak onto fresh DDO plates, and incubate at

30°C for 3–4 days.

• Short-term storage (<4 weeks): Seal with Parafilm and store at 4°C.

• Long-term storage: Scoop a large healthy colony and fully resuspend in 500 µl of YPDA

Broth + 25% glycerol (Appendix D). Store at –80°C.

NOTES:

• These diploids are useful as reference strains for checking new batches of growth media, and

for comparisons in future experiments.

• When reviving frozen stocks, remember to restreak onto DDO selective medium.

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VII. Cloning and Testing Bait for Autoactivation and Toxicity

PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING. Detailed instructions are provided to

test your bait for autoactivation (Section C) and toxicity (Section D).

A. Generate a Bait Clone

Generate a GAL4 DNA-BD fusion by cloning your gene of interest in frame with the GAL4 DNA binding

domain of the bait plasmid pGBKT7 (see Appendix C and www.takarabio.com for map).

For an extremely simple cloning procedure, we recommend using one of Takara Bio’s In-Fusion® HD

Cloning Plus Kits (see www.takarabio.com for details).

Figure 4. Simple, one-step PCR cloning with In-Fusion HD Cloning Plus Kits.

PCR Cloning of Your Bait into pGBKT7

The following method describes a simple and highly efficient method to clone your gene in-frame with

the GAL4 BD in pGBKT7. The magic of In-Fusion HD Cloning Plus (Cat. Nos. 638909, 638910,

638911 & 638920) means that:

• Your bait is automatically cloned in-frame with the bait.

• Virtually every clone contains your insert.

• It does not matter what sites are present on your bait sequence since for In-Fusion Cloning you

do not digest it.

1. Digest pGBKT7 to completion with BamHI and EcoRI, then spin column-purify.

2. Amplify your bait insert by PCR using oligos that contain a 24-bp homology to your bait, and a

15-bp homology to the linear ends of pGBKT7, which are designed as follows:

Forward Primer (111 = first codon of your bait)

5’-C ATG GAG GCC GAATTC 111 222 333 444 555 666 777 888

Reverse Primer (LLL = reverse complement of last codon of your bait)

5’-GC AGGTCGACGGATCC LLL NNN NNN NNN NNN NNN NNN NNN

NOTE: These primers actually contain 16 bp of homology in order to keep the BamHI and EcoRI

sites intact.

3. Mix the bait and linear pGBKT7 together and “fuse”’ using the In-Fusion enzyme.

See the In-Fusion HD Cloning Plus Kit User Manual at www.takarabio.com for additional details

regarding PCR cloning procedures.

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• If you wish to find proteins that interact with a membrane-bound or secreted protein, it may be

necessary to first modify the protein (van Aelst et al. 1993) or to use only selected domains as the

bait (as in Kuo et al. 1997).

• In order to confirm that the fusion construct is in-frame, the fusion junction may be sequenced

using a standard T7 primer.

B. Detecting Bait Expression

If you wish to determine whether or not your bait is expressed well in yeast, both of the following

antibodies will detect bait proteins in yeast containing pGBKT7-based bait plasmids (via Western blot).

In order to make yeast protein extracts (yeast cannot simply be boiled or sonicated to extract protein), we

strongly recommend that you use the supporting protocols provided at www.takarabio.com

• GAL4 DNA-BD Monoclonal Antibody (Cat. No. 630403)

• c-Myc Monoclonal Antibody (Cat. No. 631206)

NOTE: Use Y2HGold [pGBKT7-53] as a positive control that expresses a 57 kD protein.

C. Protocol: Testing Your Bait for Autoactivation

As a first step for any two-hybrid screen, it is imperative to confirm that your bait does not autonomously

activate the reporter genes in Y2HGold, in the absence of a prey protein.

1. Materials:

• pGBKT7 containing your gene of interest cloned in frame with the GAL4 DNA-BD

(pGBKT7).

• Competent Y2HGold cells [see Yeastmaker Yeast Transformation System 2 User Manual

(PT1172-1), supplied with this system]

• SD/–Trp/X-alpha-Gal agar plates (Appendix D)

• SD/–Trp/X-alpha-Gal/AbA agar plates (Appendix D)

NOTE: X-alpha-Gal is required, not X-Gal (Appendix D).

2. Transform 100 ng of your pGBKT7+Bait construct using the Yeastmaker Yeast Transformation

System 2 (supplied with this system).

NOTE: (POSITIVE and NEGATIVE CONTROLS) For comparison, we recommend that you

also plate the diploid controls that you created in Section VI; but plate them on DDO/X/A since

they contain both bait and prey plasmids).

3. Spread 100 µl of a 1/10 dilution and a 1/100 dilution of your transformation mixture onto

separate plates, as follows:

• SD/–Trp plates = SDO

• SD/–Trp/X-alpha-Gal = SDO/X plates

• SD/–Trp/X-alpha-Gal/AbA = SDO/X/A plates

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4. Expected results after 3–5 days:

Sample

Selective Agar Plate

Distinct 2-mm Colonies

Color

Bait autoactivation test

SDO

Yes

White

Bait autoactivation test

SDO/X

Yes

White or very

pale blue

Bait autoactivation test

SDO/X/A

N/A

Positive control

(Section VI)

DDO/X/A

Yes

Blue

NOTE: If your bait autoactivates the AbA

reporter (i.e., blue colonies appear on SDO/X/A),

check to see if it also activates the His3/Ade2 reporters by plating on SD–Trp/–His/–Ade. See

Section XI (Troubleshooting) if your bait activates all the reporters.

D. Protocol: Testing Your Bait for Toxicity

You should demonstrate that your bait protein is not toxic when expressed in yeast. If your bait is toxic to

the yeast cells, both solid and liquid cultures will grow more slowly.

If expression of your bait protein does have toxic effects, you may wish to switch to a vector (such as

pGBT9) that has a lower level of expression.

NOTE: pGBT9 is supplied as a transformation control in Takara Bio's Yeastmaker Transformation

System 2 (supplied with this system).

1. Materials:

• Y2HGold competent cells [see Yeastmaker Yeast Transformation System 2 User Manual

(PT1172-1), supplied with this system]

• SD/–Trp agar plates (Appendix D)

• SD/–Trp broth (Appendix D)

2. Transform 100 ng of the following vectors:

• pGBKT7 (empty)

• pGBKT7 + cloned bait gene

3. Spread 100 µl of 1/10 and 1/100 dilutions of your transformation mixtures onto SD/–Trp.

4. Grow at 30°C for 3–5 days:

NOTE: If your bait is toxic, you may notice that colonies containing your bait vector are

significantly smaller than colonies containing the empty pGBKT7 vector.

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VIII. Two-Hybrid Library Screening Using Yeast Mating

PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING. Detailed instructions are provided for

performing a yeast two-hybrid library screening.

• Simply mix a concentrated bait culture with 1 ml of your Mate & Plate library and incubate overnight

before plating on DDO/X/A selective media (see Figure 3).

• In addition to plating on DDO/X/A media, it is imperative that you determine the number of clones that

you screened on DDO (following the protocol below ensures this). Screening fewer than 1 million clones

may result in an inability to detect positive interactions.

With a “normalized” Mate & Plate library, up to threefold fewer clones need to be screened, because gene

representation has been equalized, significantly reducing the abundance of housekeeping genes in the library.

Several normalized libraries are available from www.takarabio.com.

1. Materials:

• Mate & Plate Library—make your own, using our Make Your Own “Mate & Plate” Library

System (Cat. No. 630490) or purchase separately.

• Bait construct transformed into Y2HGold on SD/–Trp (Section VII)

• SD/–Trp Broth (Appendix D)

• 2X YPDA Broth (Appendix D)

• 0.5X YPDA broth (Appendix D)

• kanamycin sulfate (50 mg/ml)

• YPDA + 25% glycerol [Freezing Medium] (Appendix D)

• The following selective SD agar plates (also see Appendix D):

Agar Media

Acronym

Number of Plates

SD/–Trp

—

5–10 (100 mm plates)

SD/–Leu

—

5–10 (100 mm plates)

SD/–Leu/–Trp

DDO

5–10 (100 mm plates)

SD/–Leu/–Trp/X-alpha-Gal/AbA

DDO/X/A

50–55 (150 mm plates)

SD/–Ade/–His/–Leu/–Trp/X-alpha-

Gal/AbA

QDO/X/A

5–10 (100 mm plates)

NOTE: Takara Bio’s Mate & Plate libraries are supplied in yeast strain Y187, so your bait must be in

yeast strain Y2HGold.

If you wish to “Make your Own Mate & Plate Library”, use our kit (Cat. No. 630490)

(see www.takarabio.com for details)

2. Construct your bait, test for autoactivation and toxicity (Section VII).

3. Perform the control experiments (Section VI).

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NOTE: Control experiments are strongly recommended; the control strains will aid interpretation of

results when you screen your library.

4. Prepare a concentrated overnight culture of the bait strain (Y2HGold [pGBKT7+Bait]) as follows:

a. Inoculate one fresh, large (2–3 mm) colony of your bait strain into 50 ml of SD/–Trp liquid

medium.

b. Incubate shaking (250–270 rpm) at 30°C until the OD

600

reaches 0.8 (16–20 hr).

c. Centrifuge to pellet the cells (1,000 g for 5 min), discard the supernatant.

d. Resuspend the pellet to a cell density of >1 x 10

cells per ml in SD/–Trp (4–5 ml).

[The cells can be counted using a hemocytometer.]

5. Combine the Library Strain with the Bait Strain as follows:

a. Thaw a 1-ml aliquot of your library strain in a room temperature water bath. Remove 10 µl for

titering on 100-mm SD/–Leu agar plates (see Appendix B, Section B for library titering

instructions).

NOTE: Use a hemocytometer to count the cells. Your 1 ml library aliquot should contain

>2 x 10

cells. To check the titer, spread 100 µl of 1/100, 1/1,000, 1/10,000 dilutions on SD/–Leu

agar plates. If your titer is 2 x 10

cells/ml, you will obtain 200 colonies on the 1/10,000 dilution

plate.

b. Combine 1 ml of your Mate & Plate Library with 4–5 ml Bait Strain (from Step 4) in a sterile 2-L

flask.

c. Add 45 ml of 2xYPDA liquid medium (with 50 µg/ml kanamycin).

d. Rinse cells from the library vial twice with 1 ml 2xYPDA and add to the 2-L flask.

6. Incubate at 30°C for 20–24 hr, slowly shaking (30–50 rpm).

IMPORTANT: Use the lowest shaking speed possible that prevents the cells from settling at the base of

the flask. Vigorous shaking can reduce the mating efficiency, but shaking too slowly will cause the cells

to sediment, also lowering the mating efficiency.

7. After 20 hr, check a drop of the culture under a

phase contrast microscope (40X). If zygotes are

present, continue to Step 8, if not, allow mating to

continue; incubate for an additional 4 hr.

NOTE: A zygote typically has a 3-lobed structure

(see Figure 5). The lobes represent the two haploid

parental cells and the budding diploid cell. Some

zygotes may resemble a clover leaf, while others

may take on a shape similar to a “Mickey Mouse”

face.

8. Centrifuge to pellet the cells (1,000 g for 10 min).

Figure 5. An example of a typical yeast zygote.

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9. Meanwhile, rinse the 2-L flask twice with 50 ml 0.5X YPDA (with 50 µg/ml kanamycin), combine the

rinses, and use this to resuspend the pelleted cells.

10. Centrifuge to pellet the cells (1,000g for 10 min) and discard the supernatant.

11. Resuspend all pelleted cells in 10 ml of 0.5X YPDA/Kan liquid medium. Measure the total volume of

cells + medium.

NOTE: e.g., 10 ml medium + 1.5 ml cells = 11.5 ml

12. From the mated culture, spread 100 µl of 1/10, 1/100, 1/1,000, and 1/10,000 dilutions on each of the

following 100-mm agar plates and incubate at 30°C for 3–5 days.

• SD/–Trp

• SD/–Leu

• SD/–Leu/–Trp (DDO)

NOTE: This step is essential to calculate the number of clones screened (see Step 14).

13. Plate the remainder of the culture, 200 µl per 150-mm on DDO/X/A (50–55 plates). Incubate at 30°C for

3–5 days.

14. Calculate the number of screened clones (diploids) by counting the colonies from the DDO plates after

3–5 days.

• Number of Screened Clones = cfu/ml of diploids x resuspension volume (ml)

• It is imperative that at least 1 million diploids are screened, since using less than this will result in

less chance of detecting genuine interactions on Aureobasidin A plates (DDO/X/A).

Example Calculation

• Resuspension volume (Step 11) = 11.5 ml

• Plating Volume = 100 µl

• 50 colonies grew on the 1/1,000 dilution on DDO plates.

Therefore, Number of Clones screened = 50 x 11.5 x 10 x 1,000 = 5.75 million

15. Determine the Mating Efficiency

Mating efficiencies of 2–5% are readily achieved using this procedure. If your mating efficiency is less

than 2% and you cannot screen 1 million diploids (Step 14), refer to the Troubleshooting Guide (Section

XI) for tips on improving the mating efficiency, and screen more clones.

a. Measure viabilities

• No. of cfu/ml on SD/–Leu = viability of the Prey Library

• No. of cfu/ml on SD/–Trp = viability of Bait

• No. of cfu/ml on SD/–Leu/–Trp = viability of diploids

NOTE: The strain (bait or prey) with the lower viability is the "limiting partner."

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b. Calculate Mating Efficiency (percentage of diploids):

No. of cfu/ml of diploids x 100 = % Diploids

No. of cfu/ml of limiting partner

Example Calculation

• Resuspension volume (Step 11) = 11.5 ml

• Plating Volume = 100 µl

• 5,000 colonies grew on the 1/10,000 on SD/–Trp

• 100 colonies grew on the 1/10,000 dilution on SD/–Leu

• 50 colonies grew on the 1/1,000 dilution on DDO plates

Therefore (in cfu/ml),

- Viability of Prey Library = 1 x 10

- Viability of Bait = 5 x 10

- Viability of Diploid = 5 x 10

Since the Prey Library is the limiting partner in this example, mating efficiency is calculated as

follows:

5 x 10

x 100 = 5% Mating Efficiency

1 x 10

16. Patch out all the blue colonies that grew on DDO/X/A onto higher stringency QDO/X/A agar plates using

a flat sterile toothpick or yellow pipette tip (Figure 6).

Figure 6. High stringency screening of potential interactors.

NOTE: Although it is possible to screen directly on high stringency QDO/X/A, Takara Bio recommends

screening first on lower stringency DDO/X/A to detect as many positives interactors as possible before

confirming those by patching on highest stringency plates.

17. All QDO/X/A positive interactions must be further analyzed (Section X) to identify duplicates and to

verify that the interactions are genuine.

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IX. Analysis of Results

After high-stringency patching to identify potential binding partners for your protein of interest, you may have

very few positives, or too many positives to analyze. In these scenarios, we recommend checking the following:

A. Too Few Positives

Have you screened >1 million independent clones? Refer to Section VIII, Step 14 to determine if you

screened 1 million independent clones? Optimize the mating/transformation procedure (see Section XI.

Troubleshooting Guide) and repeat the screening procedure.

• Check that your DDO/X/A and QDO/X/A growth media performs as expected with the positive

and negative controls. Very little AbA (125 ng/ml) is required, so make certain not to use too

much.

• If you screened >1 million independent clones and detected no positive colonies on DDO/X/A,

repeat the screen with a reduced concentration of Aureobasidin A (150 ng/ml instead of

200 ng/ml).

B. Too Many Positives

Have you determined that your bait does not autoactivate the reporters (Section VII.C)?

• Check that your DDO/X/A and QDO/X/A growth media performs as expected with the positive

and negative controls.

• Your bait may interact with a partner that is abundant in the library. Sort duplicates by Yeast

Colony PCR (Section X.A). After the clones have been sorted into groups, a representative of

each unique type can then be analyzed for false positive interactions (Section X.C).

• Alternatively, you may wish to try a Normalized Mate & Plate Library; see www.takarabio.com

for details.

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X. Confirmation of Positive Interactions & Rescue of the Prey Plasmid

PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING. Detailed instructions are provided for

yeast colony PCR to eliminate duplicates (Section A), rescue and isolation of library plasmids responsible for

activation of reporters (Section B), and distinguishing genuine positive from false positive interactions

(Section C).

The following represents the recommended order of events to confirm that the positive interactions are genuine.

The strategy is summarized in Figure 7. Note, however, that your preferred order of events may be somewhat

determined by the number of positives obtained from your assay. For instance, if your bait protein interacts with a

protein that is abundant in the library, you may have a large number of potential positives to sort, many of which

may be the same. In this case you may choose to perform colony PCR (Section X.A) to sort the duplicate clones

before segregating and rescuing the plasmid. If you have a low number of positive clones, you may choose to

omit the colony PCR screening step altogether and proceed directly to the Easy Yeast Plasmid Isolation Kit

(Cat. No. 630467).

We recommend performing the following steps prior to sequencing your positive clones:

• Yeast Colony PCR

• Rescue and isolation of the library plasmid responsible for activation of reporters

• Distinguishing genuine positive from false positive interactions

Figure 7. Strategies for analyzing and verifying putative positive interactions.

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A. Protocol: Yeast Colony PCR Analysis to Eliminate Duplicate Clones

1. Use the Matchmaker Insert Check PCR Mix 2 (Cat. No. 630497) to amplify your prey library

inserts. The kit includes a premix of enzyme, reagents, and primers to amplify cDNA inserts from

pGADT7 vectors. You can then characterize the inserts in Steps 2–4 using restriction enzyme analysis

to identify potential duplicates clones. We strongly recommend this complete premix because we find

that it performs very well in yeast colony samples.

2. Analyze PCR products by electrophoresis on a 0.8% TAE Agarose/EtBr gel. The presence of more

than a single band is common, indicating the presence of more than one prey plasmid in a cell.

NOTE: To confirm that similar sized bands contain the same insert, digest the PCR product with

AluI or HaeIII or another frequently cutting enzyme, and analyze the products on a 2% agarose/EtBr

gel.

3. If a high percentage of the colonies appear to contain the same AD/library insert, expand your PCR

analysis to another batch of 50 colonies.

4. At this stage, to quickly identify the clones, the PCR products (observed as a single band on gel) can

be spin column-purified and sequenced using T7 primer.

B. Protocol: Rescue and Isolation of Library Plasmid Responsible for

Activation of Reporters

1. Segregation of Library Plasmid in Yeast

Transformed yeast cells (unlike transformed E. coli cells) can harbor more than one version of a

related plasmid. This means that in addition to containing a prey vector that expresses a protein

responsible for activating the reporters, a yeast cell may also contain one or more prey plasmids that

do not express an interacting protein.

• If you rescue the plasmid via E. coli transformation without first segregating the non-

interacting prey, there is a chance that you will rescue a non-interacting prey plasmid.

• To increase the chance of rescuing the positive prey plasmid, we recommend that you streak

2–3 times on DDO/X (no Aureobasidin A), each time picking a single blue colony for

restreaking. After the first streaking, you may see a mixture of blue and white colonies,

indicating segregation of positive interactors (blue) from non-interactors (white). After

streaking one or two more times, you should only see blue colonies. The plasmid should be

rescued from one of these clones (see Step 2).

2. Rescuing the Library Plasmid from Yeast

The following methods are recommended for rescuing your plasmid from yeast:

• To identify the gene responsible for the positive interaction, rescue the plasmid from yeast

cells grown on QDO/X using the Easy Yeast Plasmid Isolation Kit (Cat. No. 630467)

• If your bait is cloned in pGBKT7 (which contains a kanamycin resistance gene), you can

select for the prey plasmid simply by selection on LB plus 100 µg/ml ampicillin using any

commonly used cloning strain of E. coli, e.g., DH5α, or Stellar™ Competent Cells (Cat. No.

636763).

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C. Protocol: Distinguishing Genuine Positive from False Positive Interactions

Y2HGold Yeast Strain contains four reporters under the control of three distinct GAL4 UAS sequences.

As a result, of following the high-stringency screening protocols described in this user manual, the

incidence of false positives is reduced to a minimum compared to other systems. The incidence of false

positives is further reduced with Normalized Mate & Plate Libraries due to more equal representation of

each transcript. However, with every two-hybrid screen there is a chance of detecting false positives and

it is important to confirm that your interactions are genuine using the following criteria (see Figure 8):

• Genuine Positive: Both Bait and Prey are required to activate the Gal4-responsive reporters

• False Positive: Prey can activate the Gal4-responsive reporters in the absence of your bait.

Figure 8. Illustration of the activation of reporter gene expression in genuine and false positives.

You can confirm protein interactions in yeast on selective media (see Appendix D for recipes) using the

following cotransformation procedure (Figure 9). This can also be done by yeast mating (see Section

VIII).

1. Materials:

• Competent Y2HGold cells [see Yeastmaker Yeast Transformation System 2 User Manual

(PT1172-1), supplied with this system]

• SD/-Leu/-Trp/X-alpha-Gal Agar (Appendix D) = DDO/X

• SD/-Ade/-His/-Leu/-Trp/X-alpha-Gal/AbA (Appendix D) = QDO/X/A

2. Using the small-scale transformation procedure, cotransform100 ng of each of the following pairs

of vectors into Y2HGold Competent Cells:

• pGBKT7/Bait + Prey (in pGADT7, pGADT7-Rec, or pGADT7-RecAB)

• Empty pGBKT7 + Prey (in pGADT7, pGADT7-Rec, or pGADT7-RecAB)

NOTE: We recommend that you perform the experiment side by side with the positive and

negative controls (Section VI).

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Figure 9. Using cotransformation on selective media to verify protein interactions. Expected results from genuine interactions.

3. Spread 100 µl of 1/10 and 1/100 dilutions of the transformation mix on the following plates:

• DDO/X

• QDO/X/A

4. Expected results after 3–5 days at 30°C (Figure 9):

a. Genuine Positive:

Sample

Selective Agar Plate

Distinct 2-mm Colonies

Color

Bait + candidate prey

DDO/X

Yes

Blue

QDO/X/A

Yes

Blue

Empty pGBKT7 + candidate prey

DDO/X

Yes

White

QDO/X/A

N/A

b. False Positive:

For false positive interactions, similar numbers of blue colonies are observed on all plates

(indicating that the prey does not require your bait to activate the reporters).

Sample

Selective Agar Plate

Distinct 2-mm Colonies

Color

Bait + candidate prey

DDO/X

Yes

Blue

QDO/X/A

Yes

Blue

Empty pGBKT7 + candidate prey

DDO/X

Yes

Blue

QDO/X/A

Yes

Blue

NOTE: Theoretically, for positive interactions, the number of colonies should be the same on both

media: DDO selects for both plasmids, and QDO/X/A selects for the plasmids as well as for the

interaction of the hybrid proteins encoded by them. However, a difference is usually observed

(10–60% lower on QDO/X/A, depending on the strength of the interaction).

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D. Sequence Analysis of a Genuine Positive

Once an interaction has been verified as being genuine, the prey insert can be identified by sequencing.

Use only DNA isolated from E. coli for this procedure. AD/library cDNA inserts can be sequenced using

the following:

• Matchmaker AD LD-Insert Screening Amplimer Set (Cat. No. 630433),

• T7 Sequencing Primer

Always check the vector sequence against the primer you wish to use. Be aware that some Matchmaker

AD plasmids (e.g., pACT2) do not contain a T7 Promoter.

Verify the presence of an open reading frame (ORF) fused in frame to the GAL4 AD sequence, and

compare the sequence to those in GenBank, EMBL, or other databases.

NOTES:

Before considering any of the following possibilities, we recommend that you verify that your clone is not

a false positive (Section X.C).

• Most genuine positive clones will activate all reporters, however it is possible that some library

clones only activate a selection of the reporters, for example the colony grows on QDO but does not

turn blue in the presence of X-alpha-Gal. This may be due to inaccessibility of a particular prey

fusion protein to a specific UAS. Confirm the interaction by additional means such as

coimmunoprecipitation.

• Most library clones will contain some 3’ untranslated region, be sure to scan the entire sequence to

find any portion of coding region fused in-frame to the GAL4 AD.

• Yeast tolerate translational frameshifts. A large ORF in the wrong reading frame may correspond to

the protein responsible for the interaction. To verify this, re-clone the insert in-frame (this can be

easily done using Takara Bio’s In-Fusion PCR Cloning Systems (see www.takarabio.com) and

determine if the AbA

, ADE2, HIS3, and MEL1 reporters are still active if your bait is also present.

• If your sequencing results reveal a very short peptide (<10 amino acids) fused to the AD—or no

fusion peptide at all—keep sequencing beyond the stop codon. You may find another (larger) open

reading frame (ORF). Such gaps can occur when a portion of the 5' untranslated region of an mRNA

is cloned along with the coding region. A Western blot using Gal4 AD Antibody (Cat. No. 630402)

will reveal the presence and size of an AD fusion protein.

• In some cases, two different ORFs may be expressed as a fusion with the AD even though a non-

translated gap comes between them. This is due to occasional translational read-through.

• If your sequencing results fail to reveal any ORF in frame with the AD coding region, it could be that

the positive library clone is transcribed in the reverse orientation from a cryptic promoter within the

ADH1 terminator on the bait plasmid (Chien et al. 1991).

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E. Biochemical Methods to Confirm Positive Interactions

We recommend confirming positive interactions using the following methods:

• After sequencing the positive clones, most researchers choose to confirm each protein-protein

interaction using independent, biochemical methods, such as affinity chromatography and/or

immunoprecipitation (Fields & Sternglanz, 1994).

• You can also test protein-protein interactions in mammalian cells using either the Matchmaker

Mammalian Two-Hybrid Assay Kit 2 (Cat. No. 630305) or the pCMV-Myc & pCMV-HA

Vector Set (Cat. No. 631604) The Vector Set includes c-Myc and HA-Tag antibodies for the

isolation and identification of protein-protein complexes.

F. Downstream Analysis

You may wish to compare the strengths of two different interactions—for example, between a bait and

two different prey proteins; or analyze the effects of point mutations on the strength of interaction, using

the following methods:

Quantitative test for interactions: The Gal-responsive LacZ gene (beta-galactosidase) integrated in

Y187 is not secreted (in contrast to alpha-galactosidase encoded by MEL1) and it cannot be used for

blue/white screening on agar plates. However, LacZ is an ideal reporter for quantitative studies of protein-

protein interactions. We recommend the use of yeast strain Y187 for such quantitative studies because the

LacZ promoter in this strain expresses strongly (Y2HGold/Y187 diploids can also be used). Quantitative

LacZ assays are described in supporting Matchmaker protocols at www.takarabio.com.

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XI. Troubleshooting Guide

Problem

Possible Explanation

Solution

DNA-BD/bait

activates reporter

genes

The bait protein has a transcriptional

activation domain. This is especially likely

if the bait protein is a transcription factor

(Ma & Ptashne, 1987; Ruden et al. 1991;

Ruden, 1992). Acidic amphipathic

domains are often responsible for

unwanted transcriptional activation

(Ruden et al. 1991; Ruden, 1992).

Remove the activating domain by creating specific

deletions within the gene. Retest the deletion

constructs for activation. At the amino acid level, a

net negative charge per 10 amino acids is a

minimal AD. Note that such deletions may also

eliminate a potentially interacting domain.

Excessive

background

• Improper media preparation

Remake SD/–Leu/–Trp/X-alpha-Gal/AbA medium.

• 0.5X YPDA medium is too rich for the

resuspension of transformed cells

Use water, TE, or 0.9% NaCl.

Low mating

efficiency

Insufficient number of pretransformed bait

cells in the mating

When you prepare the overnight liquid culture of

the bait strain, be sure to use a large, fresh colony

for the inoculum. After centrifuging and

resuspending the culture, count the cells using a

hemocytometer. The concentration should be

≥1 x 10

cells/ml, an ~100-fold excess over the

pretransformed library cells.

One or both of the hybrid proteins is toxic

to yeast

You may be able to genetically engineer the

hybrid protein in a way that will alleviate its toxicity

but still allow the interaction to occur; or use a

DNA-BD or AD vector that expresses lower levels

of the fusion protein (e.g., pBridge or pGBT9).

Bait protein is toxic to the yeast cells

•

In some cases, strains that do not grow well in

liquid culture will grow reasonably well on

agar plates. Resuspend the colony in 1 ml of

SD/–Trp, then spread the cell suspension on

five 100-mm SD/–Trp plates. Incubate the

plates at 30°C until the colonies are confluent.

Scrape the colonies from each plate, pool

them in one tube, and resuspend in a total of

5 ml of 0.5X YPDA. Use the cell suspension

in the normal mating procedure.

•

It may be necessary to perform the mating on

agar plates (Bendixen et al. 1994) or on filters

(Fromont-Racine et al. 1997). Be sure to set

up controls that will allow you to compare the

library mating efficiency with that of your bait

strain mated to Y187[pGADT7-T] and with

that of Y187[pGADT7-T] mated to

Y2HGold[pGBKT7-53].

• Bait proteins may interfere with mating if they

are highly homologous to proteins involved in

yeast mating (e.g., pheromone receptors). If

sequence information on your bait protein is

available, check it for homology to proteins

known to be involved in yeast mating (Schultz

et al. 1995; Pringle et al. 1997). In the rare

case of homology to a pheromone receptor, it

may be necessary to screen the library using

a conventional library-scale yeast

transformation.

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Problem

Possible Explanation

Solution

Failure to detect

known protein

interactions

•

If one of the following situations is

occurring, it may interfere with the

ability of the two-hybrid proteins to

interact: (1) the hybrid proteins are not

stably expressed in the host cell; (2)

the fused GAL4 domains occlude the

site of interaction; (3) the hybrid protein

folds improperly; or (4) the hybrid

protein cannot be localized to the yeast

nucleus. (See van Aelst et al. [1993] for

one example.)

Construct hybrids containing different domains of

the bait protein. For example, to study proteins

that normally do not localize to the nucleus, it may

be necessary to generate mutant forms of the

protein that can be transported across the nuclear

membrane.

•

Some types of protein interactions may

not be detectable in a GAL4-based

system.

Try using a LexA-based two-hybrid system.

•

Some protein interactions are not

detectable using any type of two-

hybrid assay.

AD/library plasmid

activates all four

reporters

independent of the

DNA-BD/bait

False positives. The AD/library hybrid

activates transcription and binds because

it recognizes the promoter regions of the

reporters.

Refer to Section X for methods to verify protein

interactions; see Serebriiskii et al. (2000) and

Bartel et al. (1993a) for further discussion of false

positives.

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XII. References

• For general reviews on yeast two-hybrid systems, see Allen et al. 1995; Bartel et al. 1993a, 1993b; Bartel & Fields,

(1997); Fields, 1993; Fields & Sternglanz, 1994; Fritz & Green, 1992; Guarente, 1993; Hopkin, 1996; Luban & Goff,

1995; McNabb & Guarente, 1996; Mendelsohn & Brent, 1994.

• An extensive list of Matchmaker System citations can be obtained from our website (

www.takarabio.com

• For additional two-hybrid references, see the Golemis lab Web Site (http://www.fccc.edu:80/research/labs/golemis) or

use MedLine (http://www.ncbi.nlm.nih.gov/PubMed/medline.html) and search under key words "two-hybrid.”

Allen, J. B., Wallberg, M. W., Edwards, M. C. & Elledge, S. J. Finding prospective partners in the library: the yeast two-

hybrid system and phage display find a match. TIBS

:511–516 (1995).

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. Current Protocols in

Molecular Biology (John Wiley & Sons, Inc.) (1994).

Bartel, P. L. & Fields, S. The Yeast Two-Hybrid System (Oxford University Press, Oxford) (1997).

Bartel, P. L., Chien, C.-T., Sternglanz, R. & Fields, S. Using the two-hybrid system to detect protein-protein interactions.

In Cellular Interactions in Development: A Practical Approach., ed. Hartley, D. A. (Oxford University Press, Oxford) pp.

153–179 (1993a).

Bartel, P. L, Chien, C.-T., Sternglanz, R. & Fields, S. Elimination of false positives that arise in using the two-hybrid system.

BioTechniques

:920–924 (1993b).

Bendixen, C., Gangloff, S. & Rothstein, R. A yeast mating-selection scheme for detection of protein-protein interactions.

Nucleic Acids Res.

:1778–1779 (1994).

Borson, N. D., Sato, W. L. & Drewes, L. R. A lock-docking oligo(dT) primer for 5’ and 3’ RACE PCR. PCR Methods Appl.

:144–148 (1992).

Chien, C. T., Bartel, P. L., Sternglanz, R. & Fields, S. The two-hybrid system: A method to identify and clone genes for

proteins that interact with a protein of interest. Proc. Nat. Acad. Sci. USA

:9578–9582 (1991).

Fields, S. The two-hybrid system to detect protein-protein interactions. METHODS: A Companion to Meth. Enzymol.

116–124 (1993).

Fields, S. & Song, O. A novel genetic system to detect protein-protein interactions. Nature

340

: 245–247 (1989).

Fields, S. & Sternglanz, R

The two-hybrid system: an assay for protein-protein interactions. Trends Genet.

: 286–292

(1994).

Fritz, C. C. & Green, M. R. Fishing for partners. Current Biol.

:403–405 (1992).

Fromont-Racine, M., Rain, J.-C. & Legrain, P. Toward a functional analysis of the yeast genome through exhaustive two-

hybrid screens. Nature Genetics

:277–282 (1997).

Giniger, E., Varnum, S. M. & Ptashne, M. Specific DNA binding GAL4, a positive regulatory protein of yeast. Cell

:767–

774 (1985).

Giniger, E. & Ptashne, M. Cooperative DNA binding of the yeast transcriptional activator GAL4. Proc. Natl. Acad. Sci.

USA

:382–386 (1988).

Golemis, E. A., Gyuris, J. & Brent, R. Analysis of protein interactions; and Interaction trap/two-hybrid systems to identify

interacting proteins. In Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), Sections 20.0 and 20.1 (1996).

Guarente, L. Strategies for the identification of interacting proteins. Proc. Natl. Acad. Sci. USA

:1639–1641 (1993).

Gubler, U. & Hoffman, B. J. A simple and very efficient method for generating cDNA libraries. Gene

:263–269 (1983).

Guthrie, C. & Fink, G. R. Guide to yeast genetics and molecular biology. In Methods in Enzymology (Academic Press, San

Diego)

194

:1–932 (1991).

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Page 31 of 41

Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. & Elledge, S. J. The p21 Cdk-interacting protein Cip1 is a potent

inhibitor of G1 cyclin-dependent kinases. Cell

:805–816 (1993).

Hopkin, K. Yeast two-hybrid systems: more than bait and fish. J. NIH Res.

:27–29 (1996).

Iwabuchi, K., Li, B., Bartel, P. & Fields, S. Use of the two-hybrid system to identify the domain of p53 involved in

oligomerization. Oncogene

:1693–1696 (1993).

Kuo, H. J., Maslen, C. L., Keene, D. R. & Glanville, R. W. Type VI collagen anchors endothelial basement membranes by

interacting with type IV collagen. J. Biol. Chem.

272:

26522–26529 (1997).

Li, B. & Fields, S. Identification of mutations in p53 that affect its binding to SV40 T antigen by using the yeast two-hybrid

system. FASEB J.

:957–963 (1993).

Luban, J. & Goff, S. P. The yeast two-hybrid system for studying protein-protein interactions. Current Opinion in

Biotechnol.

:59–64 (1995).

McNabb, D. S. & Guarente, L. Genetic and biochemical probes for protein-protein interactions. Curr. Opin. Biotechnol.

(5):554–559 (1996).

Mendelsohn, A. R. & Brent, R. Biotechnology applications of interaction traps/two-hybrid systems. Curr. Opinion in

Biotechnol.

:482–486 (1994).

Pringle, J. R., Roach, J. R. & Jones, E. W., eds. The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell

Cycle and Cell Biology (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) (1997).

Ruden, D. M. Activating regions of yeast transcription factors must have both acidic and hydrophobic amino acids.

Chromosoma

101

:342–348 (1992).

Ruden, D. M., Ma, J., Li, Y., Wood, K. & Ptashne, M. Generating yeast transcriptional activators containing no yeast protein

sequences. Nature

350

:250–251 (1991).

Schultz, J., Ferguson, B. & Sprague, Jr., G. F. Signal transduction and growth control in yeast. Curr. Opinion in Genet.

Devel.

:31–37 (1995).

Serebriiskii, I., Estojak J., Berman M. & Golemis E. A. Approaches to detecting false positives in yeast two-hybrid systems.

Biotechniques

:328–336 (2000).

van Aelst, L., Barr, M., Marcus, S., Polverino, A. & Wigler, M. Complex formation between RAS and RAF and other

protein kinases. Proc. Natl. Acad. Sci. USA 90:6213–6217 (1993).

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Appendix A: Mate & Plate Library Construction

A. Host Strain Information

The phenotypes and complete genotypes of Y2HGold and Y187 (the library strain) are shown in Tables II

and III. For additional information on the growth and maintenance of yeast, see the supporting

Matchmaker protocols at www.takarabio.com. We also recommend the Guide to Yeast Genetics and

Molecular Biology (Guthrie & Fink, 1991).

Table II. Yeast Host Strain Genotypes

Strain

Genotype

Reporters

Transformation

Markers

Reference

Y2HGold

b, d

MATa, trp1-901, leu2-3, 112,

ura3-52, his3-200, gal4Δ, gal80Δ,

LYS2 : : GAL1

UAS

–Gal1

TATA

–His3,

GAL2

UAS

–Gal2

TATA

–Ade2

URA3 : : MEL1

UAS

–Mel1

TATA

AUR1-C MEL1

AbA

HIS3,

ADE2, MEL1

trp1, leu2

Nguyen,

unpublished

Y187

MATα, ura3-52, his3-200,

ade2-101, trp1-901, leu2-3, 112,

gal4Δ, gal80Δ, met–,

URA3 : : GAL1

UAS

–Gal1

TATA

–LacZ,

MEL1

MEL1, LacZ

trp1, leu2

Harper

et al. 1993

The GAL1, GAL2, and MEL1 upstream activating sequences (UASs) are recognized and bound by the Gal4 BD. The trp1, his3,

gal4, and gal80 mutations are all deletions; leu2-3, 112 is a double mutation.

Y2HGold is a derivative of strain PJ69-2A (James et al. 1996). The ade2-101 mutation in the precursor strain, PJ69-2A, was

replaced (by recombination) with the GAL2–Ade2 reporter construct. In the absence of GAL4, Y2HGold displays the Ade–

phenotype.

The LacZ reporter construct was integrated into the yeast genome by homologous recombination at the ura3-52 mutation (A. Holtz,

unpublished). Recombinants were selected on SD/–Ura. The met– phenotype in this strain is unstable.

The AUR1-C reporter construct was integrated into the yeast genome by homologous recombination at the ura3-52 mutation (Y.

Nguyen, unpublished). Recombinants were selected on SD/–Ura. The met– phenotype in this strain is unstable.

Table III. Phenotype Testing on Various SD Media

Strain

SD/–Ade

SD/–His

SD/–Leu

SD/–Trp

SD/–Ura

Y2HGold

–

Y187

–

Y2HGold

[pGBKT7-53]

–

Y187 [pGADT7-

–

Control Diploid

Diploid strain derived from mating Y2HGold [pGBKT7-53] with Y187 [pGADT7-T].

B. General Considerations for Mate & Plate Library Construction

Matchmaker Libraries may be cloned into one of several commonly used Gal4 AD vectors. Three of these

are described below. To find out which vector your library was constructed in, refer to the Certificate of

Analysis (CofA) included with your library. Vector maps are available at www.takarabio.com.

• Libraries constructed in pGADT7-Rec

Mate & Plate Libraries cloned in pGADT7-Rec are constructed using our Make Your Own “Mate &

Plate” Library System (Cat. No. 630490). pGADT7-Rec offers a simple and efficient method for

constructing two-hybrid libraries via recombination-mediated cloning in vivo directly in S. cerevisiae. See

the Make Your Own “Mate & Plate” Library System User Manual (PT4085-1) at www.takarabio.com

for a description of the procedure.

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• Libraries constructed in pGADT7-RecAB

Normalized Mate & Plate libraries are constructed in pGADT7-RecAB. cDNA synthesized using our

SMART® technology is normalized to reduce the proportion of highly abundant transcripts. Normalized

cDNA is SfiI-digested, and cloned into the SfiI A/B sites of pGADT7-RecAB.

Once cloned, the library is amplified in E. coli, rescued, and used to transform yeast strain Y187. The

resulting colonies are harvested at high density in freezing medium and immediately aliquoted and frozen

at –70°C.

• Libraries Constructed in pACT2

Mate & Plate Libraries that are cloned in pACT2 were prepared using a modified Gubler & Hoffman

procedure (1983). Once cloned, the library is amplified in E. coli. The library plasmid is then isolated and

used to transform yeast strain Y187. The resulting colonies are harvested, aliquoted, and frozen at –70°C.

C. Library Priming

Matchmaker cDNA libraries are oligo(dT)-primed, or oligo(dT) + random-primed, as stated on the

Certificate of Analysis (CofA); please refer to your CofA for the specific type of oligo(dT) primer used in

the first-strand synthesis. The “lock-docking” oligo(dT)25d(A/C/G) primer contains a degenerate

nucleotide site that positions the primer at the junction of the poly-A tail and the transcript proper (Borson

et al. 1992). This primer eliminates synthesis of lengthy poly(dT) regions and thereby enhances the

representation of full-length clones and 3' ends in the library (Borson et al. 1992). Random priming may

lead to a greater representation of all portions of the gene, including amino-terminal and internal domains,

regardless of mRNA secondary structure; random priming also generates a wider size-range of cDNA.

For oligo(dT) + random-primed libraries, separate first-strand syntheses are performed with each type of

primer; after second-strand synthesis (before ligation to the adaptor), the cDNAs are pooled in roughly

equal proportions.

Unidirectional libraries are made with oligo(dT) primers that have one vector-compatible restriction

enzyme site. The other site is added (with sticky ends) by the adaptor that is ligated to the cDNA. Thus,

digestion with one restriction enzyme ensures the cDNA’s proper orientation when ligated to a vector that

has been digested with the appropriate two enzymes.

D. Adaptors and Linkers

Please refer to the Product Certificate of Analysis (CofA) for information on the adaptor or linker used in

the construction of your Matchmaker Library. pACT2-based libraries are made using adaptor ligation.

pGADT7-Rec- and pGADT7-RecAB-based libraries are constructed using Takara Bio’s SMART

technology, as outlined in the user manual of the ”Make Your Own Mate & Plate Library System” (Cat.

No. 630490)

NOTES:

• The open reading frame of the insert starts at the codon immediately following the C-terminal codon

(amino acid 881) of the GAL4 AD, not within the adaptor.

• If an EcoRI linker is used, the cDNA is methylated to protect any internal EcoRI sites.

• If an adaptor is used in the construction of nondirectionally cloned libraries, no methylation or

restriction enzyme digestion of the cDNA is required; therefore, any internal EcoRI sites present in

the cDNA will not be cut.

• If an adaptor is used in the construction of unidirectionally cloned libraries, the cDNA is methylated

to protect the alternative site.

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• If the library is synthesized using EcoRI/NotI/SalI adaptors, you may excise the inserts from the

vector using sites within the adaptor.

• For information about pGADT7-Rec, please refer to Vector Information Packet PT3530-5, supplied

with all libraries constructed in this vector.

E. cDNA Size Fractionation

All ds cDNA is size-fractionated to remove unincorporated primers, unligated adaptors, and adaptor

dimers; this process also removes low-molecular-weight (i.e., <400 bp) incomplete cDNAs. Matchmaker

Libraries have a wide range of insert sizes (generally >400 bp), which may be an advantage in a two-

hybrid library screening (Fritz & Green, 1992; Fields & Sternglanz, 1994). See the CofA for quality

control information about insert size.

F. Normalized Mate & Plate Libraries

Please refer to the CofA for information on the normalization procedure used in the construction of the

Normalized Libraries.

G. Insert Size Range and Average Insert Size

Sizes are determined by running the cDNA on a gel prior to cloning, and comparing the profile to MW

size markers.

H. Library Amplification

Unless otherwise stated on the CofA, libraries constructed in pACT2 and pGADT7-RecAB were

amplified once in E. coli. Libraries constructed in pGADT7-Rec are not amplified since they are made

directly in yeast.

I. Quality Control of the Original cDNA Library

• cDNA size range and average cDNA size are determined by running the cDNA on a gel prior to

cloning and comparing the profile to MW size markers. The average insert size range is 0.5 kb to

4.0 kb.

• Number of independent clones is estimated before amplification. All libraries are guaranteed to

have at least 1 x 10

independent clones and are representative of the mRNA population

complexity. See Ausubel et al. (1994) for a general discussion of library size.

J. Quality Control for the Mate & Plate Libraries

Refer to the CofA included with your library.

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Appendix B: Library Titering

A. General Considerations

• Diluted libraries are always less stable than undiluted libraries. Therefore, once the library

dilutions are made, plate them within the next hour, before misleading reductions in titer can

occur.

• Use proper sterile technique when aliquoting and handling libraries.

• Design and use appropriate controls to test for cross-contamination.

• Always use the recommended concentration of antibiotic (i.e., kanamycin) in the medium to

suppress growth of contaminating bacteria.

B. Library Titering

1. Materials:

• YPDA Broth (Appendix D)

• SD/–Leu (100-mm plates) (Appendix D)

• Sterile glass spreading rod, bent Pasteur pipette, or 5-mm glass beads for spreading

culture on plates.

2. Transfer the 10-µl library aliquot (reserved from Section VIII) to 1 ml of YPDA Broth in a 1.5-ml

microcentrifuge tube. Mix by gentle vortexing. This is Dilution A (dilution factor = 10

–2

NOTE: Matchmaker Gold Libraries are extremely viscous, so pay special attention during

aliquoting.

3. Remove 10 µl from Dilution A and add it to 1 ml of YPDA Broth in a 1.5-ml microcentrifuge

tube. Mix by gentle vortexing. This is Dilution B (dilution factor = 10

–4

4. Add 10 µl from Dilution A to 50 µl of YPDA Broth in a 1.5-ml microcentrifuge tube. Mix by

gentle vortexing. Spread the entire mixture onto an SD/–Leu plate.

5. Remove 50 µl aliquots from Dilution B and spread onto separate SD/–Leu plates as above.

6. Invert the plates and incubate at 30°C for 3–5 days.

NOTE: Colony size will vary, depending on the insert.

7. Count the number of colonies on plates having 30–300 colonies.

8. Calculate the titer (cfu/ml) as follows:

Number of colonies = cfu/ml

plating volume (ml) x dilution factor

NOTE: Due to slight variability in pipettes and pipetting techniques, a 2–5-fold range in titer

calculations is not unusual.

Example calculation

• No. of colonies on plate = 100

• Plating volume = 0.05 ml

• Dilution factor = 10

–4

100 = 2 x 10

cfu/ml

0.05 ml x 10

-4

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Figure 10. Map of pGBKT7-53 DNA-BD control plasmid. pGBKT7-53 is a positive control plasmid that encodes a fusion of

the murine p53 protein (a.a. 72–390) and the GAL4 DNA-BD (a.a. 1–147). The murine p53 cDNA (GenBank Accession No.

K01700) was cloned into pGBKT7 at the EcoRI and BamHI sites. The p53 insert was derived from the plasmid described in

Iwabuchi et al. (1993); plasmid modification was performed at Takara Bio. pGBKT7-53 has not been sequenced.

Figure 11. Map of pGADT7-T AD control plasmid. pGADT7-T is a positive control plasmid that encodes a fusion of the SV40

large T antigen (a.a. 87–708) and the GAL4 AD (a.a. 768–881). The SV40 large T antigen cDNA (GenBank Locus SV4CG),

which was derived from the plasmid referenced in Li & Fields (1993), was cloned into pGADT7; plasmid modification was

performed at Takara Bio. pGADT7-T has not been sequenced and it is not known whether any of the sites are unique.

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Appendix D: Yeast Growth Media & Supplements

A. Ready-to-go Media Pouches Available from Takara Bio

Takara Bio offers media sets with a complete assortment of mixes in convenient, “ready-mixed” foil

pouches, for use with the Matchmaker Gold Yeast Two-Hybrid System. We also provide the media

supplements Aureobasidin A and X-alpha-Gal in an optional combination with the media set. In addition,

each of the media mixes as well as these two supplements may be purchased separately.

• See Table IV for a list of the components of the Yeast Media Set 2 (Cat. No. 630494) and the

Yeast Media Set 2 Plus (Cat. No. 630495). The Yeast Media Set 2 Plus also contains the

additional media supplements Aureobasidin A and X-alpha-Gal, which are required for the

protocols described in this user manual.

• See Table V for information for purchasing each of the media mixes separately, in packs of 10

pouches.

• See Table VI for information on preparing all additional required media supplements and

purchasing Aureobasidin A and X-alpha-Gal separately.

Table IV. Components of Yeast Media Set 2 & Yeast Media Set 2 Plus

Media Pouch

Quantity of Pouches Supplied

Volume of Media Each Pouch Makes

YPDA Broth

0.5 L

YPDA with Agar

0.5 L

SD/–Leu Broth

0.5 L

SD/–Leu with Agar

0.5 L

SD/–Trp Broth

0.5 L

SD/–Trp with Agar

0.5 L

SD/–Leu/–Trp with Agar

0.5 L

SD/–Ade/–His/–Leu/–Trp with Agar

0.5 L

Additional Components in Yeast Media Set 2 Plus

X-alpha-Gal

250 mg

—

Aureobasidin A

1 mg

—

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Table V. Individual Yeast Media Pouches for Matchmaker Gold Protocols

Yeast Media Pouches

Takara Bio

Cat. No.

Quantity of

Pouches

Supplied

Volume of Media

Each Pouch

Makes

Rich Media (for Routine Culturing of Untransformed Yeast)

YPDA Broth

YPDA with Agar

630306

630307

0.5 L

Minimal Media Single Dropouts (SDO)

SD–Trp Broth

SD–Trp with Agar

SD–Leu Broth

SD–Leu with Agar

630308

630309

630310

630311

0.5 L

Minimal Media Double Dropouts (DDO)

SD–Leu/–Trp Broth

SD–Leu/–Trp with Agar

630316

630317

0.5 L

Minimal Media Triple Dropouts (TDO)

SD–His/–Leu/–Trp Broth

SD–His/–Leu/–Trp with Agar

630318

630319

0.5 L

Minimal Media Quadruple Dropouts (QDO)

SD–Ade/–His/–Leu/–Trp Broth

SD–Ade/–His/–Leu/–Trp with Agar

630322

630323

0.5 L

Table VI. Additional Media & Media Supplements Required for a Two-Hybrid Screen

Freezing Medium

Preparation Instructions

YPDA Medium & 25% glycerol

See Section E of Appendix D

Supplement Name

Takara Bio Cat. No.

Stock Solution Concentration

Aureobasidin A

630466

630499

500 µg/ml (see Section F of Appendix D)

X-alpha-Gal (250 mg)

630462

630463

20 mg/ml in dimethyl formamide

Kanamycin sulfate

—

50 mg/ml stock solution

Dimethyl formamide

—

Unless otherwise specified.

B. General Media Preparation Instructions

• Prepare media by dissolving pouch contents in 500 ml ddH

O, autoclave for 15 min at 121˚C, and

allow to cool before use (or filter-sterilize broth media). Do not over-autoclave.

• This media does not usually require pH adjustment, but if your source water is particularly acidic,

you may need to adjust the pH of the media to 5.8.

• For additional information on preparing media from the pouches, please see the Takara Bio Yeast

Media Protocol-at-a-Glance (PT4057-2) at www.takarabio.com

C. 2X YPDA Broth

Reconstitute one YPDA Broth pouch in 250 ml ddH

O and sterilize.

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D. 0.5X YPDA Broth

Reconstitute one YPDA Broth pouch in 1 L ddH

O and sterilize.

E. Freezing Medium

Mix 100 ml YPDA (sterile) and 50 ml 75% glycerol (sterile).

F. Aureobasidin A Stock Solution Recipe:

Dissolve 1 mg Aureobasidin A (Cat. No. 630466) in 2 ml of absolute ethanol for a stock concentration of

500 µg/ml. Store at 4°C.

G. Aureobasidin A Working Concentration

Add just 200 µl of the Aureobasidin A stock solution to 500 ml of dropout agar media for yeast two-

hybrid screening, yielding a final concentration of 200 ng/ml.

H. X-alpha-Gal Stock Solution Recipe

Dissolve X-alpha-Gal (Cat. No. 630463) at 20 mg/ml in dimethylformamide (DMF). Store X-alpha-Gal

solutions at –20°C in the dark.

I. X-alpha-Gal Working Concentration

Add 1 ml of the X-alpha-Gal stock solution to 500 ml dropout media for yeast two-hybrid screening.

J. SDO Agar Plates

Single dropout (–Trp) or (–Leu) media is prepared as follows:

• Prepare 500 ml agar media using SD/–Trp (or SD/–Leu) with Agar, autoclave, and cool to

55–60°C in a water bath.

• Mix, pour immediately, and allow to dry.

K. SDO/X Agar Plates

Single dropout (–Trp) media containing 40 µg/ml X-alpha-Gal is prepared as follows:

• Prepare 500 ml agar media using SD/–Trp with Agar, autoclave, and cool to 55–60°C in a water

bath.

• Add 1 ml of X-alpha-Gal Stock Solution.

• Mix, pour immediately, and allow to dry.

L. SDO/X/A Agar Plates

Single dropout (–Trp) media containing 40 µg/ml X-alpha-Gal and 200 ng/ml Aureobasidin A is prepared

as follows:

• Prepare 500 ml agar media using SD/–Trp with Agar, autoclave, and cool to 55–60°C in a water

bath.

• Add 1 ml of X-alpha-Gal Stock Solution.

• Add 200 µl of Aureobasidin A stock solution.

• Mix, pour immediately, and allow to dry.

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M. DDO Agar Plates

Double dropout media is prepared as follows:

• Prepare 500 ml agar media using SD–Leu/–Trp with Agar, autoclave, and cool to 55–60°C in a

water bath.

• Mix, pour immediately, and allow to dry.

N. DDO/X Agar Plates

Double dropout media containing 40 µg/ml X-alpha-Gal is prepared as follows:

• Prepare 500 ml agar media using SD–Leu/–Trp with Agar, autoclave, and cool to 55–60°C in a

water bath.

• Add 1 ml of X-alpha-Gal Stock Solution.

• Mix, pour immediately, and allow to dry.

O. DDO/X/A Agar Plates

Double dropout media containing 40 µg/ml X-alpha-Gal and 200 ng/ml Aureobasidin A is prepared as

follows:

• Prepare 500 ml agar media using SD–Leu/–Trp with Agar, autoclave, and cool to 55–60°C in a

water bath.

• Add 1 ml of X-alpha-Gal Stock Solution.

• Add 200 µl of Aureobasidin A stock solution.

• Mix, pour immediately, and allow to dry.

P. QDO/X Agar Plates

Quadruple dropout media containing 40 µg/ml X-alpha-Gal is prepared as follows:

• Prepare 500 ml agar media using SD/–Ade/–His/–Leu/–Trp with Agar, autoclave, and cool to 55–

60°C in a waterbath.

• Add 1 ml of X-alpha-Gal Stock Solution.

• Mix, pour immediately, and allow to dry.

Q. QDO/X/A Agar Plates

Quadruple dropout media containing 40 µg/ml X-alpha-Gal and 200 ng/ml Aureobasidin A is prepared as

follows:

• Prepare 500 ml agar media using SD/–Ade/–His/–Leu/–Trp with Agar, autoclave, and cool to 55–

60°C in a waterbath.

• Add 200 µl of Aureobasidin A stock solution.

• Add 1 ml of X-alpha-Gal Stock Solution.

• Mix, pour immediately, and allow to dry.

R. Kanamycin Supplement to Yeast Media

Kanamycin sulfate can be added to all yeast media at a final concentration of 50 µg/ml to stop bacterial

contamination. Please note that kanamycin does not stop contaminating fungal growth, so proper sterile

technique must still be used. Also note that this antibiotic does not select for any plasmids in yeast.

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Page 41 of 41

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