Laboratory Exercise to Measure Plasmid Copy Number by qPCR

Abstract

Quantitative PCR (qPCR) has numerous applications in biology. In an education 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 ( C T ) 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.

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“…Primer sets were confirmed to have similar efficiencies (>98%) using a dilution series of purified gDNA. Copy number (CN) was determined by relative comparison of the cycle threshold (Ct) values for each target using the following equation: CN = 2 −Ct ahp -Ct rpoB [ 24 , 25 ].…”