Journal: Nature protocols
Article Title: Easi-CRISPR protocol for creating knock-in and conditional knockout mouse models using long ssDNA donors
Figure Lengend Snippet: Schematic of iv TRT and ssDNA preparation steps. A dsDNA template can be a PCR product, or a plasmid with a suitable restriction enzyme (RE) site distal to the insertion cassette (Steps 1–2) . The gel on the right shows a plasmid digested with a RE. RNA is synthesized using in vitro transcription (Steps 3–18) . The gel on the right shows an RNA of ~ 900 bases long. cDNA is synthesized by reverse transcription using a reverse primer (Steps 19–23) . The gel on the right shows a sample ssDNA (cDNA). Note that the cDNA preparation typically runs like a smear with a prominent band within the smear. Purification of ssDNA (Steps 24–35) . The image on the left shows the gel after excision of the prominent band (for purification) and the gel on the right shows the purified ssDNA. The GeneRuler DNA Ladder Mix (ThermoFisher Scientific, cat. no. SM0331) was used in all the gels as a DNA size marker.
Article Snippet: 28) Extract DNA from the gel slice using NucleoSpin Gel or PCR Clean-up (TaKaRa) kit (option A) or by Phenol extraction and ethanol precipitation (option B).
Techniques: Polymerase Chain Reaction, Plasmid Preparation, Synthesized, In Vitro, Purification, Marker