When genotyping your edited samples, we recommend performing PCR (polymerase chain reaction) to amplify the region around the edit and then proceed with Sanger sequencing.
To confirm the success and specificity of your PCR reaction, we recommend using gel electrophoresis (agarose gel) to determine the approximate size of the PCR product and to confirm PCR specificity.
If you do not observe good amplification (no visible bands) or you observe non-specific amplification (>1 strong band in your unedited control sample) we recommend the following troubleshooting steps:
- Review primer design: Primers should be unique in the genome. Confirm that the right primers for the targeted region were used, particularly when no amplification is observed.
- Confirm that your PCR conditions (cycling conditions, primer and template concentrations, etc.) are consistent with the recommendations for your PCR reagents.
- Design and test additional primers. Primer design guidance may be found in Synthego’s Genotyping Protocol.
- Perform a gradient PCR to determine the best annealing temperature for your specific experimental system.
- Contact your Taq polymerase or PCR reagent provider for product-specific troubleshooting assistance.
Posted: August 22, 2023, Last Updated: October 10, 2024