Yeast Interaction trap cloning

A very detailed 1995 technical review on two-hyb system (draft manuscript chaptor in DNA Cloning 2: A Practical Approach, Expression Systems", 2nd Edition, eds D. M. Glover and B. D. Hames. Oxford University Press, 1995). This page is maintained by Finley's Lab at Wayne state University.

Content
1. Introduction
1.1 Background
1.2 The interaction trap
2. Making and testing baits
2.1 LexA fusion expression plasmids
2.2 Reporters and yeast strains
2.2.1 LEU2 reporter strains
2.2.2 lacZ reporters
2.3 Testing the bait protein
2.3.1 Testing whether the bait protein activates transcription of the
reporters

Protocol 1. Testing baits for transcription activation

2.3.2 Demonstrating that the bait enters the yeast nucleus and binds
operators
Protocol 2. The repression assay
2.3.3 Verifying that a full-length fusion protein is made.

3. Libraries
4. An interactor hunt
4.1 Introducing the library into the selection strain.
4.1.1 Selecting interactors from library transformants.
4.1.2 Performing a one step selection for interactors

Protocol 3. Transforming the selection strain with library DNA.

4.2 Isolating yeast with galactose dependent Leu+ and lacZ+
phenotypes

Protocol 4. Selecting interactors.

5. Verifying specificity

Protocol 5. Isolating and classifying library plasmids.
Protocol 6. Determining specificity of interactors.

6. Using a mating assay to verify specificity.
Figure 4a. Mating assay cartoon.
Figure 4b. Mating assay result.

Protocol 7. Mating assay.

7. Expected results.

Appendix
Sequencing and PCR primers for pEG202 and pJG4-5
Media recipes.

Last update 16-Mar-2005, Rating Excellent! of 2 votes.