Lab 8 Background

Yeast Genomic DNA Isolation

In order to express phage from the correctly assembled phage vector, the vector has to be purified from yeast cells. This can be done using either homemade or commercially available yeast genome extraction kit.

The procedure includes pelleting down yeast cells, re-suspending and breaking down cells using buffers, precipitating cell debris and proteins, and extracting genomic DNA.

Working with Bacterial Cells

Bacteria are single-celled, ubiquitous prokaryotic organisms that are an important part of the ecosystem. Some bacterial species are not harmful or even beneficial to humans, but many are disease-causing. The bacterial species you will be working with in this course are not harmful to humans.

Similar to yeasts, bacteria can be cultured in a liquid media or on a solid agar plate. We will do both in this course. Unlike yeasts that grow well at 30 °C, the types of bacteria we work with grow the best at 37 °C.

Phage Procurement via Cell Lysis

To test the virulence of the mutant phage in later experiments, a reasonable amount of phage needs to be procured. One of the most common ways to procure phage is through bacterial infection and cell lysis. This method takes advantage of the lytic cycle that is characteristic of the T7 phage. In short, a vector containing the mutant phage will be introduced into bacterial cells via transformation. Once in the bacterial cells, T7 phage genes will be expressed and replicated, and the resulted T7 phage viral particles (also called virions) will leave the bacterial cells by lysing (killing) them. To procure phages, we will collect the bacteria cultures after lysing occurs (viral particles will be in the media).

Reference