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. 2015 Oct 14;5:15096. doi: 10.1038/srep15096

Figure 1. General outline of the no-SCAR method.

Figure 1

On day 1 the pCas9cr4 plasmid is used to transform E. coli, followed by plating on LB + Cm, and growth at 37 °C. On day 2 the resulting strain can be transformed with pKDsg-xxx plasmid, where –xxx denotes the targeted gene, plated on LB + Spec and Cm, and incubated at 30 °C overnight. On day 3 the resulting strain is grown in SOB until OD ~0.5 and λ-red is induced with 50 mM L-arabinose. After 15–20 minutes the cells are made electrocompetent and transformed with ssDNA or dsDNA that confers a mutation to the protospacer or PAM sequence. After 1–2 hours of recovery the cells are plated on LB + Spec, Cm, and aTc, then incubated at 30 °C overnight. On day 4 colonies are screened by PCR and grown at 37 °C to cure the pKDsg-xxx plasmid. The next pKDsg-xxx plasmid is then used to transform the mutant strain on Day 5 and the process is repeated.