λ dna hindiii digest

Genomic DNA was extracted from 500 µl of anticoagulated peripheral blood using a Flexigene DNA Kit (Qiagen, Hilden, Germany) and diluted to a final volume of 25 μl with double-distilled water. The quality and quantity of DNA were assessed using a Denovix spectrophotometer and by 1% agarose gel electrophoresis. CNBP expanded alleles were detected as previously described (Nakamori et al., 2009 (link)), with modifications. Briefly, 2 μg of genomic DNA was digested with AluI and HaeIII and the fragments were resolved by 0.4% agarose gel electrophoresis at 40 V for 40 hr. After denaturation and neutralization, the DNA was transferred to a nylon membrane (MilliporeSigma, Burlington, MA) and fixed by UV cross-linking using a Stratalinker 2400 (Stratagene, San Diego, CA). The membrane was hybridized for 16 hr at 65°C with a digoxigenin (DIG)-labeled locked nucleic acid (LNA) probe (CCTG)₅ at a concentration of 10 pmol/ml. After washing at high stringency, the signal was revealed using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Basel, Switzerland) and visualized using an ImageQuant LAS 4000 device (GE Healthcare, Chicago, IL). Bands were sized by running two sets of molecular weight markers alongside the samples: DNA Molecular Weight Marker XV (Expand DNA Molecular Weight Marker, Roche) and λ DNA-HindIII Digest (New England Biolabs, Ipswich, MA).

The amplification products were analyzed by 2.0% agarose gel electrophoresis, using EtBr as a fluorescent dye (0.5 µg/mL, final), and visualized/documented with a GelDoc™ EZ Imager (Biorad). The 100-bp DNA Ladder and λ-DNA/HindIII Digest (New England Biolabs) were the DNA markers used to assess molecular weights. The amplicons: 628 bp for rpsL, 645 bp for rrs and 719 bp for gidB, were purified using a QIAquick PCR Purification Kit, as recommended by the manufacturer (Qiagen).

Standard molecular weight markers were prepared by dephosphorylating 17 μg λ DNA-HindIII Digest (New England Biolabs N3012S) with 10 U Antarctic Phosphatase (New England Biolabs M0289S) in total volume of 40 μL for 1 h at 37°C. The phosphatase was then inactivated by incubation at 80°C for 10 min. Subsequently, 6.8 μg of dephosphorylated DNA was labeled with γ-[³²P]-ATP using 40 units of T4 Polynucleotide Kinase (New England Biolabs M0201S) for 1 h at 37°C, in a total reaction volume of 40 μl. Unincorporated γ-[³²P]-ATP was removed using Illustra MicroSpin G-50 columns (GE Healthcare) and 5 mM EDTA was added to the recovered sample.
For Figure 1D, end-labeled 3189 bp plasmid was prepared by digesting 4 μg plasmid DNA with 2 μL SmaI (Roche) in 1X CutSmart Buffer (New England Biolabs) in a 40 μL final reaction volume for 2 h at 25°C. The linearized plasmid was then column purified using the High Pure PCR Product Purification Kit (Roche) and then dephosphorylated and end-labeled with γ-[³²P]-ATP, as described for standard molecular weight markers. Other labeled markers in Figure 1D were generated by PCR amplification using oligonucleotides 7272 – 7275 (see Table S2) and pTDK13 plasmid template. In each case, 50 μL PCR reactions were assembled in the presence of 33 nM α-[32P]-dCTP and the PCR products were purified over Illustra MicroSpin G-50 columns (GE Healthcare).