no-insert, pSpCas9(BB)-only negative control for ligation. Note: if you are using the Cas9 D10A nickase mutant for subsequent applications, substitute pSpCas9(BB) with pSpCas9n(BB). Alternatively, if fluorescence-based screening or selection is needed, substitute with pSpCas9(BB)-2A-GFP, pSpCas9(BB)-2A-Puro, pSpCas9n(BB)-2A-GFP or pSpCas9n(BB)-2A-Puro. The following steps use pSpCas9(BB) as an example: ComponentsAmount (μl)pSpCas9(BB), 100 ng×Diluted oligo duplex from Step 5B(iii)2Tango buffer, 10×2DTT, 10 mM1ATP, 10 mM1FastDigest BbsI1T7 ligase0.5ddH2Oto 20Total20Incubate the ligation reaction for a total of 1 h. Cycle numberCondition1-637 °C for 5 min, 21 °C for 5 minTreat the ligation reaction with PlasmidSafe exonuclease to digest any residual linearized DNA. This step is optional but highly recommended. ComponentAmount (μl)Ligation reaction from Step 5B(v)11PlasmidSafe buffer, 10×1.5ATP, 10 mM1.5PlasmidSafe exonuclease1Total15Incubate the PlasmidSafe reaction at 37 °C for 30 min, followed by 70 °C for 30 min.
