Phosphor imaging screen

To determine the presence of the (CTG⋅CAG)500 repeat in CRISPR/Cas9 genome-edited DM500 myoblasts, we used PCR amplification of the (CTG⋅CAG)500 repeat-containing gene segment, followed by Southern blot hybridization. PCR products were resolved by electrophoresis on a 1% agarose gel, transferred by capillary transfer to a Hybond-XL nylon membrane (Amersham Pharmacia Biotech), and hybridized with ³²P-labeled oligonucleotides. A DMPK oligo (5′-AGAACTGTCTTCGACTCCGGG-3′), located 5′ of the CRISPR cleavage sites, was used to visualize PCR products from both the unmodified DMPK gene and from cells with a deletion of the region between the two CRISPR sites. The oligo was 5′ end labeled using ɣ³²P-ATP and T4 polynucleotide kinase and hybridized to the membrane in Church-Gilbert hybridization solution overnight at 42°C. The membrane was washed, exposed to a Bio-Rad Phosphor Imaging Screen, and imaged by Phosphor-Imager analysis (Molecular Imager FX, Bio-Rad). Analysis was performed with Quantity One (Bio-Rad) and ImageJ software. After imaging, the probe was stripped from the membrane using boiling buffer (0.1 X SSC and 0.1% SDS). Subsequently, using similar conditions for hybridization, washing, and exposure analysis, ³²P-end-labeled (CAG)9 probe was used to visualize PCR products containing an expanded (CTG⋅CAG)n repeat.