Agarose gel dna extraction kit

The 147-bp 601 nucleosome positioning DNA sequence^{28 (link)} was modified to introduce a single THF group 11 bp from the 5′ end of the J chain, which is equivalent to 64 nt from the dyad, and designated as THF (+64). The forward primer containing the THF: 5′-FAM-CAC AGG ATG TTHFGA TAT CTG GCC TGG AGA CTA G-3′, the reverse primer: 5′-TGG AGA ATC CCG GTG CCG AGG CCG CTC AAT TG-3′, and the plasmid: pGEM-3Z/601 were used to generate the substrate via the polymerase chain reaction (PCR) listed in Table S1. Following a PCR reaction, the PCR product was concentrated using a standard ethanol precipitation protocol and purified from a 1.2% agarose gel using a DNA agarose gel extraction kit (Qiagen). For the substrates containing the 5S nucleosomal positioning sequence^{21 (link)}, the 162-bp DNA substrate was generated by first ligating the damaged strand containing a single THF group and 5′-FAM, using a splinter complementary DNA upstream and downstream of the lesion. This ligation reaction contained 110 units of E. coli DNA ligase and 1x ligation buffer provided by the manufacturer (New England Biolabs). The ligated product and the undamaged complementary strand (162-mer) were PAGE purified and subsequently annealed by heating to 95°C for 10 min and slow cooling in buffer containing 30 mM Tris, pH 7.5 and 100 mM potassium acetate. The dsDNA substrate is listed in Table S1.