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 and ICE-D 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. 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.
Articles in this section
- How do I use the ICE analysis tool and interpret my results?
- How many samples can ICE analyze at once?
- What RNA-guided nucleases does ICE analysis support?
- What size PCR amplicons should I use for ICE and ICE-D analysis tool? How should I design my PCR primers?
- Are there parameters that I can specify, such as indel window or expected cutting site?
- Can I run ICE on a private server or in the cloud?