Materials

Buffers and Solutions

Chloroform A

Ethanol

Isopropanol

Phenol (equilibrated to pH 8.0) A

STES lysis solution O

Yeast lysis solution C )

Yeast rescue buffer i )

Enzymes and Buffers

Restriction endonucleases EcoRI, XhoI, HaeIII Media

CM selective medium

Use the table in Chapter 18, Protocol 1 Stage 1 to prepare the necessary selective media. LB agar plates containing 50 pg/ml ampicillin O Minimal (-trp) medium for bacteria Yeast selective X-gal medium £ ✓

Additional Reagents

Step 2 of this protocol requires the reagents for electroporation listed in Chapter 1, Protocol 26.

Steps 3 and 4 of this protocol require the reagents for the preparation of miniprep DNA from plasmids listed in

Chapter 1, Protocol 1.

Step 10 of this protocol requires the reagents for sequencing of yeast DNA listed in Chapter 4, Protocol 13. Vectors and Bacterial and Yeast Strains

E. coli DH5« or E. coli KC8 (pyrF leuB600 trpC hisB463; CLONTECH), competent for electroporation

Please see Chapter 1, Protocol 26.

Nonspecific bait plasmid

Please see the note to Step 6.

pMW112, pRFHM-1

pBait (from Chapter 18, Protocol 1 Stage 1)

Yeast colonies with the appropriate phenotype growing on a CM(Glu)-Ura-His-Trp master plate (identified in Chapter 18, Protocol 2 Stage 2, Step 21)

Yeast strain EGY48

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