? TROUBLESHOOTING 116Day 3: isolate the plasmid DNA from overnight cultures by using a QIAprep spin miniprep kit.117Sanger sequencing. Verify the sequence of each colony by sequencing from the pUC19 backbone using the pUC19-Fwd or pUC19-Rev primer. Reference the sequencing results against the expected genomic sequence to check for the presence of Cas9-induced NHEJ or HDR modifications. Calculate the percentage of editing efficiency as (no. of modified clones)/ (no. of total clones). It is important to pick a reasonable number of clones (>24) to generate an accurate approximation of modification efficiencies.
