Summary of project:

Project Objective: to increase the capacity of University of Zimbabwe students and staff in the application of basic molecular biology methods applicable to many scientific disciplines.

 

What are the students working on?

The teaching lab focuses on the production and utilization of the enzyme Taq DNA polymerase. 

 

Protocol for producing Taq DNA polymerase:

A culture with 250 ml LB broth and 100µl of glycerol stock containing Taq-expressing plasmid, pAKTaq, provided by Addgene (see references below) is incubated in a water bath at 37°C.

 

The culture is grown until turbid; a colorimeter can be used to determine when A600 = 0.6-0.8, which can take several hours.

 

Taq polymerase gene expression is induced by adding IPTG to 0.2 mM (50 µl of 1 M IPTG in 250 ml culture).

 

The culture is induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG).

 

After the 16 hours of growth overnight, the culture is placed on ice, then dispensed into microcentrifuge tubes and centrifuged at 4,300 rpm for 5 minutes.

 

The supernatant is discarded and the pellet taken up in 200 μl TEN buffer (10 mM Tris HCl, pH 8.0; 1 mM EDTA, and 100 mM NaCl) and the mixture heat inactivated for 30 min at 75 °C.

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1 ml of the end product is pipetted into a microcentrifuge tube and kept at 4°C in a bench top cooler for use, or it can be frozen for storage.

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Addgene is a US-based non-profit plasmid repository whose mission is to accelerate research and discovery by improving access to useful research materials and information. Addgene facilitates the sharing of high-quality scientific materials, research reproducibility, and open science by archiving and distributing DNA-based research reagents and associated data to scientists worldwide.