PCR-based screening of small plasmid inserts
Year: 2020
Type: article
Abstract: Plasmid construction typically uses DNA ligase and/or extraction of product from gels. These methods often require troubleshooting, and low transformation rate can lead to excessive time and resources spent screening candidates. Additionally, small inserts can be difficult or impossible to distinguish by size-separation through gel electrophoresis, and may not contain enzyme restriction sites. Single guide RNA (sgRNA), frequently used in CRISPR-Cas9 gene editing, contains a universal Cas9 binding scaffold (tracrRNA) fused to a 17 – 20-nt target-specific guide (crRNA) and can be expressed from a plasmid. Due to its specificity, the crRNA must be replaced for each unique target. Here, we used a tracrRNA-encoding plasmid as a template and added the crRNA-encoding sequence to demonstrate a fast and effective method of identifying small inserts in plasmids using PCR. This approach allows researchers to narrow down candidates to as few as one sample before confirming through sequencing.
Source: PubMed Central
Institution: Syracuse University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3006899993