▲ CRITICAL Instead of the SURVEYOR or RFLP assays, genomic amplicons of the target region (produced in Step 78 or 101) from transfected cells (Steps 8–13, and 14–28 for HEK 293FT cells, or steps 43–51 for HUES9 cells) can be cloned into a plasmid, and a set of clones can be prepared for Sanger sequencing to assess Cas9 cleavage or HDR-mediated target modification efficiency. SURVEYOR or HDR primers can be used for Sanger sequencing if appropriate restriction sites are appended to the forward and reverse primers. For cloning into the recommended pUC19 backbone, EcoRI can be used for the Fwd primer and HindIII for the Rev primer. 109Target-site amplicon digestion. Set up the digestion reaction as follows: ComponentAmount (μl)FastDigest buffer, 10×3FastDigest EcoRI1FastDigest HindIII1Purified PCR product, 20 ng μl−110(Step 78 or 101)ddH2O15Total volume30110pUC19 backbone digestion. Set up the digestion reaction as follows and incubate it at 37 °C for 15 min: ComponentAmount (μl)FastDigest buffer, 10×3FastDigest EcoRI1FastDigest HindIII1FastAP alkaline phosphatase1pUC19 vector (200 ng μl−1)5ddH2O20Total volume30111Purify the digestion reactions with the QIAQuick PCR purification kit.
