PCR amplicons of an sgRNA targeted genomic region should be generated in a similar way to those used for TIDE analysis. In general, a 400-800 bp PCR amplicon length is best amenable to ICE analysis. For best results, the use of PCR primers that are 18–22 bp in length, with Tm >55°C and 45–60% GC content should be used. It is best to design the forward and reverse primers at least 150 bp from the closest sgRNA cut-site to allow for optimal sequencing across the edit. There are a number of free online tools which can help you with designing primers such as Primer 3. Tools such as Primer Blast can also be used to check for off-target amplification.
The genomic PCR conditions including primers, annealing temperature, and amount of genomic DNA need to be optimized until a single band of the correct size is obtained. After DNA extraction from a cell population, PCR amplicons should be verified by gel electrophoresis and purified prior to Sanger sequencing.