Exo sap treated

Genomic DNA was isolated from Can^R clones using a previously described method⁶⁴ . Cells were resuspended in 1.5 µL of 0.5 mg/mL lyticase solution [0.9 M Sorbitol, 0.1 M EDTA (pH 7.4)] in microplates, incubated at 37°C for 15 min, then resuspended in 50 µL of water. The samples were incubated at 100°C for 5 min and centrifuged at 2,500 × g for at least 2 min. For PCR analysis of CAG repeat length, reactions included 1× Green GoTaq reaction buffer (M7911, Promega), 0.16 mM dNTP mix, 0.8 µM of each primer, 0.5 units of Taq DNA polymerase (SibEnzyme or Thermo Scientific), and 1 µL of the DNA supernatant in a 12.5 µL total reaction volume. Primers JK316 and JK317 result in a 544 bp product for (CAG)₁₄₀. Primers JK318 and JK319 result in a 625 bp product for (CAG)₁₄₀ (Figure 1B). Primers JK153 and JK171 result in a 462 bp product for (CAG)₁₄₀ and 321 bp (CAG)₉₃. 10 µL PCR products were run on 1.5% agarose in 0.5× TBE alongside 50 bp and 100 bp DNA ladders (NEB). PCR products were sized using TotalLab Quant software for 1D DNA gels.
The same PCR method was used to generate products for sequencing the CAG repeats (FlankL and CanF) or the CAN1 gene (JK167 and JK168, JK169 and JK170). PCR products were Exo-SAP treated (Affymetrix) and sequenced by Eton Bioscience or University of Chicago Sequencing Core).