
Laboratory Exercise to Measure Plasmid Copy Number by qPCR
Author(s) -
Benjamin David,
Jinbei Li,
Faisal Masood,
Caroline M. Blassick,
Paul A. Jensen,
Karin Jensen
Publication year - 2021
Publication title -
journal of microbiology and biology education
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.301
H-Index - 7
eISSN - 1935-7885
pISSN - 1935-7877
DOI - 10.1128/jmbe.00125-21
Subject(s) - plasmid , computational biology , troubleshooting , transformation (genetics) , biology , real time polymerase chain reaction , gene , computer science , genetics , operating system
Quantitative 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 ( 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. (This work was presented at the 2020 Biomedical Engineering Society annual meeting).