Illumina MiSeq paired end sequencing; Sequence Requirements for Cascade Function in a Type I-E CRISPR System: Plasmid Loss
Sequence Requirements for Cascade Function in a Type I-E CRISPR System: Plasmid Loss
• Start a 2 mL overnight for each Plasmid loss strain ( 13 different protospacer plasmids) in LB + Kan and amp at 37C, 225rpm. • Subculture all strains together into 10 mL of LB (12.5 L for 8 protospacers and 7.7 L for 13 protospacers) • Make electrocomp cells • Electroporate in pBAD33 and pBAD33-Cas3 WT into strains • Plate onto M9 + 0.2% glycerol + 0.2% arabinose + chloramphenicol carbenicillin • Next day: Scrape plates into 1.5 mL of LB, spin down, dump supernatant, and can freeze at -20 C here.  pelleted cells were used as the source of DNA for PCR amplification o PCR protospacer with ultramers using a tiny bit of cells 40 L Taq Rxn, 68 anneal, 30 sec extension• Fwd: 1:10 dilution of 8567• Rev: 1:100 dilution of 8556-8566 and 8537