C9ORF72 Repeat Expansion Analysis by Southern Blot

Southern blot analysis was used to analyze the C9ORF72 repeat expansion mutation as described^{69 (link)}. DNA was isolated using Wizard® genomic DNA purification kit (Promega). 20 µg of DNA was digested overnight with Xba endonuclease, separated on a 0.8% agarose gel electrophoresis, gel was subsequently depurinated, denatured, and transferred overnight on to a positively charged nylon membrane (Roche). The membrane was UV cross-linked and hybridized overnight at 47 °C with a digoxigenin (DIG)-labeled PCR probe. PCR probe was synthesized from genomic DNA using PCR DIG probe synthesis kit (Roche) using following primers South Fw: 5′-CTTGCAGATCAAAAGGCACA-3′ and South Rev: 5′-TTGACGCACCTCTCTTTCCT-3′. Following hybridization, membrane was washed and labeled with anti-DIG antibody and developed using CDP-Star reagent as per manufacturer’s recommendation (Roche).