? TROUBLESHOOTING 101Purify the PCRs by using the QIAQuick PCR purification kit.102In the HDR example provided in Figure 6, a HindIII restriction site was inserted into the EMX1 gene. These are detected by an RFLP analysis of the PCR amplicon: ComponentAmount (μl)Purified PCR ampliconx (200-300 ng)FastDigest buffer1Hindlll (or other enzyme as necessary)0.5ddH2OUp to 10Total10103Digest the DNA for 10 min at 37 °C.104Run 10 μl of the digested product with loading dye on a 4–20% gradient polyacrylamide TBE gel until the xylene cyanol band has migrated to the bottom of the gel.105Stain the gel with SYBR Gold dye while rocking for 15 min. Be sure to shield the staining solution from light to avoid photobleaching of the dye.106Image and quantify the cleavage products as described above for the SURVEYOR assay section (Steps 86–89).107HDR efficiency is estimated by using the following formula: (b + c)/(a + b + c), where a is the integrated intensity for the undigested HDR PCR product, and b and c are the integrated intensities for the HindIII-cut fragments.108Alternatively, clone the genotype-purified PCR amplicons from Step 101 via Sanger sequencing (Steps 109–117) or deep sequencing (Steps 118–126).
