Screening a cDNA Library

for use with HybriZAP zebrafish cDNA libraries

Objective

cDNA library screening allows detection of expressed genes for subsequent cloning and sequencing.

Selecting a Library

Currently, our lab has access to cDNA libraries from three developmental time points.

• Maternal: messages from embryos before midblastula-transistion, when zygotic transcription begins (~0-3 hours of development).

• Shield: messages from the shield stage, which signifies the beginning of gastrulation (~6 hours of development).

• Post-somitegenesis: messages after the formation of somites is complete (~24 hours of development).

Choose the appropriate library for when you expect your message of interest is expressed.

Overview of Procedures

1. Titer the library to determine the concentration of infectious phage.

2. Plate and lift the phage particles.

3. Hybridize the immobilized cDNA with labeled probe.

4. Pick positive plaques.

5. Cycle through steps 2-4 until a single cDNA containing phage is isolated, generally three cycles.