Protocol

Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Storage, Maintenance, and Working with Living Arrays

This protocol was adapted from "Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array," contributed by Tony R. Hazbun and John P. Miller, Chapter 37, in Protein-Protein Interaction, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

The most difficult aspect of using an array approach for a high-throughput study is maintaining the array’s viability while avoiding contamination of the plates. This protocol describes three different storage methods for the array. Frozen stocks are the most permanent method and are an absolute requirement for maintaining a record of the array. Unfortunately, because thawing is necessary to access the strains from a 96-well format, the repeated use of the frozen stocks as a source is expected to eventually lead to loss of viability of the stock. (Generating frozen stocks of the array strains individually, as well as in the arrayed format, is essential.) Maintenance of a water stock provides a source that is more convenient and (relative to yeast cells in media) semi-permanent (~6 months). However, the water stock is fairly precarious because cross-transfer of strains between wells can occur quite easily if the plates are dropped or disturbed. The third method of storage, that of maintaining extra copies of the solid medium array plates, is the most convenient, but it is also the least stable. In short, the best option is to make several frozen stocks. For greater convenience in repeated use of the array, generate water stocks and additional copies of the solid medium plates.