Unless otherwise stated, cells were transfected in 1× Tb-BSF buffer [30 (link)] using one pulse with program X-001 in the Amaxa Nucleofector IIb (Lonza).
For calculation of recombination rate, 107L. mex Cas9 cells were transfected with 105 molecules of respective DNA fragment per cell and 40 µg in vitro transcribed RNA. Cells were recovered in M199 for 8 h, then blasticidin was added and cells were plated on a 96-well plate. The mean rate of resistance from three independent transfections was calculated from the positive wells on plates seeded with 5 × 106 cells and 5 × 105 cells.
For double tagging of PF16, 107L. mex Cas9 cells were co-transfected with 3 µg of donor DNA and 30 µg in vitro transcribed sgRNA for single tags or with 5 µg of each donor DNA and 100 µg sgRNA for double tagging.
For tests of in vivo sgRNA transcription, L. mex Cas9 T7 cells were transfected with 4 µg donor DNA and 4 µg sgRNA template DNA. Transfections with the Amaxa Nucleofector IIb used 3 × 106 cells. For transfections with the BTX ECM 830 square wave electroporation system, 107 cells were pulsed three times with 1.5 kV (unipolar, 100 µs, 10s interval) in 0.4 cm gap Gene Pulser electroporation cuvettes (BioRad).
For gene tagging with pPLOT constructs, 3 × 106 cells were transfected with the PCR reactions for the sgRNA and donor DNA (combined volume approx. 50 µl) in a total volume of 250 µl. For gene KOs with pT constructs, 107 cells were transfected with the PCR reactions for the two sgRNAs and two donor DNAs (combined volume approx. 100 µl) in a total volume of 250 µl.
For T. brucei gene tagging, PCR products were ethanol precipitated, DNA pellets resuspended in a total volume of 100 µl Tb-BSF buffer and mixed with 107 cells in 100 µl Tb-BSF buffer.
Electroporated cells were immediately transferred into pre-warmed medium and left to recover for 4–16 h before adding selection drugs. Survival of drug-resistant transfectants typically became apparent 5–7 days after transfection.
For calculation of recombination rate, 107L. mex Cas9 cells were transfected with 105 molecules of respective DNA fragment per cell and 40 µg in vitro transcribed RNA. Cells were recovered in M199 for 8 h, then blasticidin was added and cells were plated on a 96-well plate. The mean rate of resistance from three independent transfections was calculated from the positive wells on plates seeded with 5 × 106 cells and 5 × 105 cells.
For double tagging of PF16, 107L. mex Cas9 cells were co-transfected with 3 µg of donor DNA and 30 µg in vitro transcribed sgRNA for single tags or with 5 µg of each donor DNA and 100 µg sgRNA for double tagging.
For tests of in vivo sgRNA transcription, L. mex Cas9 T7 cells were transfected with 4 µg donor DNA and 4 µg sgRNA template DNA. Transfections with the Amaxa Nucleofector IIb used 3 × 106 cells. For transfections with the BTX ECM 830 square wave electroporation system, 107 cells were pulsed three times with 1.5 kV (unipolar, 100 µs, 10s interval) in 0.4 cm gap Gene Pulser electroporation cuvettes (BioRad).
For gene tagging with pPLOT constructs, 3 × 106 cells were transfected with the PCR reactions for the sgRNA and donor DNA (combined volume approx. 50 µl) in a total volume of 250 µl. For gene KOs with pT constructs, 107 cells were transfected with the PCR reactions for the two sgRNAs and two donor DNAs (combined volume approx. 100 µl) in a total volume of 250 µl.
For T. brucei gene tagging, PCR products were ethanol precipitated, DNA pellets resuspended in a total volume of 100 µl Tb-BSF buffer and mixed with 107 cells in 100 µl Tb-BSF buffer.
Electroporated cells were immediately transferred into pre-warmed medium and left to recover for 4–16 h before adding selection drugs. Survival of drug-resistant transfectants typically became apparent 5–7 days after transfection.
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