Qiaxcel advanced gel electrophoresis system

The Surveyor assay allows the detection of mutations and polymorphisms in DNA. If two strands of DNA form a heteroduplex due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions, the Surveyor nuclease recognizes the mismatch and cleaves the DNA. The cleaved DNA products can be detected by gel electrophoresis (36 (link)).
S. cerevisiae BY418 cells harboring pCascade, pCRISPR, and pCas3-Cse1 or negative controls lacking pCRISPR, pCascade, or both were grown in SC medium with or without the galactose inducer and transformed with pTargetHigh. The transformed cells were grown for 24 h in SC medium, pelleted by centrifugation, and grown again in equal volume of fresh medium for 24 h. A total of 1.5 mL of the culture was pelleted by centrifugation and resuspended in 500 μL of distilled water. The cells were lysed by heating at 98°C for 10 min, and 0.5 μL of the lysate was used as the template for PCR with primers DR003 and DR004 to amplify the target region. The PCR products were analyzed by electrophoresis on a 2% agarose gel and used for mutation detection with a Surveyor mutation detection kit (IDT) as per the manufacturer’s instructions. The result of the Surveyor assay was analyzed by using the Qiagen QIAxcel advanced gel electrophoresis system.

Probands from Families 3, 4, and 5 were identified through TaqMan assay screening and confirmed by a duplex PCR method. The additional homozygotes in the abovementioned families were only genotyped by duplex PCR. PCR was performed using the KAPA2G PCR Kit (Kapa Biosystems) and was run on the program 94 °C for 60 s followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 40 s at 72 °C. Gel electrophoresis was performed on the QIAxcel Advanced gel electrophoresis system (Qiagen). PCR primers were designed using the Primer3 program (http://bioinfo.ut.ee/primer3-0.4.0/primer3/).