Failsafe enzyme mix

STELA was performed as described previously (Baird et al. 2003 (link); Sfeir et al. 2005 (link)). Briefly, 2 µg of genomic DNA was digested with EcoRI (New England Biolabs) overnight at 37°C. Ten nanograms of digested DNA was ligated to telorettes in 10 µL of ligation buffer (5 U of T4 DNA ligase [New England Biolabs], 1× ligase buffer [New England Biolabs], 0.18 nM individual telorettes) overnight at 35°C. Ligated DNA (250 pg) was amplified by PCR in 25 µL of PCR mix (0.2 µM XpYpE2 primer, 0.2 µM teltail primer, 1× Fail Safe PCR buffer H, 2 U of Fail Safe enzyme mix [Epicentre] with 27 cycles [15 sec at 95°C, 20 sec at 58°C, and 9 min at 68°C]). PCR products were resolved on a 0.7% agarose/TAE gel, blotted onto Hybond (GE Healthcare), UV cross-linked in a Stratalinker, prehybridized with Church mix (0.5 M sodium phosphate buffer at pH 7.2, 1 mM EDTA, 0.7% SDS, 0.1% BSA), and hybridized overnight at 55°C with Klenow [α-³²P]dCTP-labeled XpYp probe in Church mix. Membranes were rinsed three times for 15 min each in Church wash (40 mM sodium phosphate buffer at pH 7.2, 1 mM EDTA, 1% [w/v] SDS) at 55°C and exposed to PhosphorImager screens.