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Fig. 4 | BMC Biotechnology

Fig. 4

From: Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein

Fig. 4

Creation of TRP3 specific Cas9-sgRNA ribonucleoprotein complex. a Amplifying the plasmid PTRB025 with primer pair NTRB107/108 to change the protospacer region. Recirculate the DNA fragment with a Mutagenesis Kit. b Amplification of the modified plasmid in E. coli and verification of the modification by sequencing with M13 primers. c Linearize of the plasmid with HindIII. d. Amplification of the sgRNA template with primer pair NTRB80/81. e Transcription of the sgRNA with T7 RNA polymerase. f DNAse treatment and RNA purification. g Cas9-sgRNA ribonucleoprotein complex formation

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