Phire green hot start 2 mastermix

T1 plants were screened for mutations using a T7 endonuclease 1 (T7E1) assay. DNA was extracted from 3-week-old T1 plants by heating a 1 mm leaf disk in 25 μL Extract-N-Amp extraction solution (E7526) at 95 °C for 10 minutes and then adding 25 μL PCR Diluent (E8155, MilliporeSigma, St. Louis, MO). Primers were selected to amplify an approximately 1000 bp region flanking the Cas9 target site (Supplemental Table S1). PCR was carried out using Phire Green Hot Start II Mastermix (ThermoFisher Scientific, Waltham, MA) under manufacturer-recommended conditions. For the T7E1 assay, the Alt-R Genome Editing Detection Kit (Integrated DNA Technologies, Coralville, IA) was used according to manufacturer specifications. In any samples with the presence of non-wildtype amplicons, PCR products were purified (Wizard SV Gel and PCR Clean-Up System, Promega Corporation, Madison, WI) and sent for Sanger sequencing at the Cornell Biotechnology Resource Center (Cornell University, Ithaca, NY). T2 seeds collected from plants with confirmed target site mutations were screened for the absence of fluorescence and the presence of a homozygous mutation at the target site using Sanger sequencing. T3 seeds collected from these non-transgenic, homozygous mutant plants were used for further analyses.

T1 plants were screened for mutations using a T7 endonuclease 1 (T7E1) assay. DNA was extracted from 3-week-old T1 plants by heating a 1 mm leaf disk in 25 μL Extract-N-Amp extraction solution (E7526) at 95 °C for 10 minutes and then adding 25 μL PCR Diluent (E8155, MilliporeSigma, St. Louis, MO). Primers were selected to amplify an approximately 1000 bp region flanking the Cas9 target site (Supplemental Table S1). PCR was carried out using Phire Green Hot Start II Mastermix (ThermoFisher Scientific, Waltham, MA) under manufacturer-recommended conditions. For the T7E1 assay, the Alt-R Genome Editing Detection Kit (Integrated DNA Technologies, Coralville, IA) was used according to manufacturer specifications. In any samples with the presence of non-wildtype amplicons, PCR products were purified (Wizard SV Gel and PCR Clean-Up System, Promega Corporation, Madison, WI) and sent for Sanger sequencing at the Cornell Biotechnology Resource Center (Cornell University, Ithaca, NY). T2 seeds collected from plants with confirmed target site mutations were screened for the absence of fluorescence and the presence of a homozygous mutation at the target site using Sanger sequencing. T3 seeds collected from these non-transgenic, homozygous mutant plants were used for further analyses.