Difference between revisions of "Team:Munich/Protocols"

For our project we used different methods which are described in respective protocols. We also included a detailed list of all chemicals, materials and the aim of each experiment. This page is for everybody who wants to reproduce our work or wants to get an overview, but also to share our experiences with the iGEM community.

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.