CRISPR-B™ vector construction
Carrying Cas9 nuclease and Red recombinase
Carrying donor template
Carrying target gene gRNA
Electroporation and CRISPR-B™ _CR competent cells preparation
1. CRISPR-B _CR vector transfer into bacteria by electroporation. Validate the transfection by colony PCR and sequencing.
2. Select the CRISPR-B _CR positive strains and prepare the CRISPR-B _CR competent cells
Transfer of CRISPR-B_G and CRISPR-B _D
Transfer vectors CRISPR-B_G and CRISPR-B_D into CRISPR-B _CR competent cells by electroporation. CRISPR and Red recombination system worked together to edit the bacterial genome.
Validate the single clones by PCR and sequencing. Eliminate CRISPR-B™ plasmids, and obtain the knockout clones.
1. Colony PCR 2.Sequencing