TY  - GEN
T1  - PCR-based screening of small plasmid inserts
AU  - Sullenberger, Matthew T
AU  - Kelley, Leanne H
AU  - Maine, Eleanor M
DO  - 10.17912/micropub.biology.000221
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000221/
AB  - 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.
PY  - 2020
JO  - microPublication Biology
ER  -