GSM6125023: Donor1 G472 RNP only - [CD5-NGS-revision-VVPlate-1C02_S11]; Homo sapiens; OTHER
5 days after electroporations, genomic DNA was isolated from each sample using DNA QuickExtract (Lucigen, Cat#QE09050) based on manufacturer protocols. Primers were designed around the guideRNA target site and included adapter sequences. Amplicons were generated by adding 0.5 µL each of forward and reverse primer at 100 µM to 10 µL of QuickExtract, 50 µL of KAPA HiFi HotStart Ready Mix PCR kit (Roche, Cat# 07958935001), and H2O to a total of 100 uL reaction volume. The following PCR thermocycler conditions were followed: 95°C for 3 minutes, 30 cycles of 98°C for 30 seconds followed by 68°C for 30 seconds and 72°C for 30 seconds, and a final 3 minute extension at 72°C. Amplicons were submitted to the Innovative Genomics Institute (IGI) Next Generation Sequencing Core for second PCR reactions and sequencing.