Cloning
We used different approaches to assemble genetic parts dependent on their design. Golden gate cloning was chosen for assembly of Cas13a-His-SUMO-Lwa into pSB1C3. We digested and ligated DNA parts of aeBlue to assemble this construct. For Intein-Extein we finally used a combination of different cloning methods to assembly the DNA fragments.
We amplified DNA fragments with standard PCR approaches. We used Q5, Phusion and Taq polymerases. Cells were transformed by chemical or electro transformation and outgrown at 37 °C for 1 h in SOC medium before plating on selective LB agar plates. We isolated plasmid DNA with the help of a commercial available kit and always eluted with nuclease-free water. With analytical restriction digest, colony PCR and agarose gels we checked the success of our cloning work. Final glycerol cryo stocks were always verified by sequencing.
|
