Team:Munich/Protocols

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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. General Cloning Cas13a Readout Other Agarose gel electrophoresis PCR Minipreparation Restriction digest Ligation Glycerol stock Ni-NTA agarose protein purification Äkta protein purification SDS PAGE RNase activity Lyophilization Expression of Cas13a and TEV Protease Phenol/Chlorophorm purification of RNA Phenol/Chlorophorm purification of genomic DNA Denaturing Urea PAGE In vitro Transcription RNA purification with Norgen Kit FINA method for RNA extraction FINA method for DNA extraction First strand cDNA syntesis RPA RPA on paper RPA TX Chemical transformation Electro transformation Chemically competent cells Electro competent cells Dephosphorylation Golden Gate Assembly Gibson Assembly PCR clean-up Sequencing sample preparation Soft litography AuNP preparation AuNP-DNA conjugation AuNP linkage AuNP cleavage Protein and nucleic acid sequences Proteins Nucleic acids 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.