Laboratory Exercise to Measure Plasmid Copy Number by qPCR.

Benjamin David,Jinbei Li,Paul Jensen,Caroline Blassick,Karin Jensen,Faisal Masood

doi:10.1128/jmbe.00125-21

Abstract

ABSTRACTQuantitative PCR (qPCR) has numerous applications in biology. In an educational setting, qPCR provides students an opportunity to better understand the PCR mechanism by providing both quantitative information about the reactions and also data to troubleshoot PCRs (e.g., melt curves). Here, we present a relatively short (2-h) laboratory activity to demonstrate qPCR to quantify plasmid copy number (CN) by measuring the cycle threshold (CT) values for a genomic gene and a plasmid gene using transformed cells as a template. The activity can be combined with additional laboratory exercises, including bacterial transformation, to create the template to be used in the qPCRs. This lab activity is ideal for undergraduate laboratory courses that include recombinant DNA technology.(This work was presented at the 2020 Biomedical Engineering Society annual meeting).

Highlights

PROCEDUREPCR is one of the most foundational tools in molecular bioengineering and has become indispensable in many disease diagnostic methods, such as COVID-19 testing (1)
The template used for this experiment is E. coli DH5α cells transformed with pGLO plasmid (Bio-Rad)
The laboratory exercise described has been implemented for seven semesters, with approximately 300 students completing the activity

Summary

PROCEDURE

PCR is one of the most foundational tools in molecular bioengineering and has become indispensable in many disease diagnostic methods, such as COVID-19 testing (1). We describe a 2-h lab exercise using qPCR to quantify CN of the pGLO plasmid in Escherichia coli strain DH5α. Cycle thresholds (CT) are measured for both a chromosomal and plasmid gene, which are used to calculate the plasmid CN This exercise addresses some limitations to using qPCR in lab courses by reducing the time, complexity, and cost necessary to complete the activity. The template used for this experiment is E. coli DH5α cells transformed with pGLO plasmid (Bio-Rad). One colony of E. coli DH5α cells transformed with the pGLO plasmid is picked and suspended in 25 ml of water as the template. The following reactions are set up in triplicate for the genomic gene and plasmid gene separately: water, forward and reverse primers, qPCR Luna mix, and template.

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RESULTS

DISCUSSION