Gene amplification by PCR and subcloning into a GFP‐fusion plasmid expression vector as a molecular biology laboratory course*

Abstract

A novel experimental sequence for the advanced undergraduate laboratory course has been developed at Earlham College. Utilizing recent improvements in molecular techniques for a time-sensitive environment, undergraduates were able to create a chimera of a selected gene and green fluorescent protein (GFP) in a bacterial expression plasmid over the course of a single semester in a weekly 3-h laboratory period. Students designed PCR primers for amplification of the selected gene using computational DNA sequence analysis tools. During the experimental portion of the course, students amplified and ligated the target DNA into a commercially available GFP expression vector. Following transformation of the ligation product, plasmids were harvested from the resulting bacterial colonies and were analyzed by restriction digestion to confirm the creation of the chimeric GFP-DNA. This course gave students valuable experience with commonly used molecular techniques in an authentic research project. In addition, students gained experience with experimental design and execution. The techniques presented here are flexible and can be generalized for use with almost any DNA sequence and expression vector. This series also serves as an example of how faculty can adapt their ongoing research projects to the undergraduate laboratory.

An in silico DNA cloning experiment for the biochemistry laboratory

“…Other instructors have reported their use of these techniques with undergraduates in the ''wet'' laboratory to clone the Oryzacystatin I gene from rice into the pET28a expression vector using the restriction enzymes NdeI and EcoRI and to ligate a target DNA into a GFP expression vector [17,18]. The in silico laboratory could be easily adapted to another protein of interest to complement other instructors' laboratory emphases.…”

A web‐based adaptive tutor to teach PCR primer design

“…By using adaptive e-learning, we added a new activity to the existing spectrum of activities that teach about PCR, such as often reported laboratory courses, (see for instance [15][16][17]) and much less reported computerbased tutorials [18].…”

From gene mutation to protein characterization

“…Because of its natural green color, this member of the green fluorescent protein family is an ideal protein to use in an undergraduate laboratory setting [3][4][5]. The green colored protein allows students (i) to visually monitor the expression of the protein in a recombinant host such as E. coli, (ii) to watch in real time the whereabouts of the protein during the purification process, and (iii) to easily characterize the protein with standard equipment found in a typical undergraduate laboratory [6,7].…”