IDT primer and gBlock ordering, hydration and aliquots SOP

Authors: Condon, Justine; Durso, Lisa

Submitted to: Protocols.io
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/13/2024
Publication Date: 12/13/2024
Citation: Condon, J.C., Durso, L.M. 2024. IDT primer and gBlock ordering, hydration and aliquots SOP. Protocols.io. https://doi.org/10.17504/protocols.io.e6nvw1r77lmk/v1.
DOI: https://doi.org/10.17504/protocols.io.e6nvw1r77lmk/v1

Interpretive Summary: One way to examine the DNA of bacteria is to use a procedure called PCR (polymerase chain reaction), which makes many copies of the DNA. Primers are used to identify where to start and stop making copies - allowing the amplification of a very specific target gene. For PCR to work, it requires not only the correct primers, but also a very precise amount of primers. A second important element of PCR is having a reliable positive control. This previously meant having a living bacterium that was positive for the gene of interest. Now it is common to use gBlocks, which are made in a process similar to primers, and which function as a positive control without the use of living bacteria. This protocol describes the standard operating procedure for all stages of primer and gBlock ordering, mixing, and storage, in support of PCR.

Technical Abstract: PCR primers and gBlock controls are an essential ingredient for the molecular characterization of bacterial isolates and environmental samples, including feces, soil, and water. This protocol describes the correct procedures on how to order primers and gBlocks, how to prepare them to a 100µM concentration, and how to aliquot them for future PCR applications.