Seaplaque gtg agarose

A fibroblast cell line generated from sixteen male Siberian tigers with normal karyotype was obtained using primary explanting techniques and cell cryogenic preservation technology [23 (link)]. The cell line was tested for viability, microorganism contamination, and chromosome euploidy according to Yasui et al. [24 (link)]. White blood cells and fibroblast cells from an adult male Siberian tiger were collected independently and mixed with 1% (w/v) Seaplaque GTG agarose (Cambrex, Rockland, ME, USA) at a concentration of 5 × 10⁷ cells/mL. The cell-agarose suspension was transferred into DNA plug molds to form solid agarose plugs.
The agarose plugs were treated with freshly prepared proteinase K digestion and then partially digested with HindIII restriction endonuclease (New England Biolabs, Baverly, MA, USA) as described in published protocols [25 (link)]. Using Lambda Ladder PFG Markers (New England Biolabs, Baverly, MA, USA) as a standard, the digested DNA plugs were subject to PFGE and the gel block containing 100–400 kb restriction fragments were cut in 0.5 cm slices. A second PFGE was then performed to remove small DNA fragments coiled within the large DNA fragments in the gel slices. The HMW DNAs were purified through electroelution and dialysis, and quantified by agarose gel electrophoresis with the λ DNA marker of known concentration.

DNA plugs were prepared from both mating types (Kaneko4: plus; Kaneko3: minus) in 1% SeaPlaque GTG agarose (Cambrex Bio Science Rockland), treated with Pronase E (Sigma), and washed thoroughly as described (Ferris et al. 2010 (link)). BAC library production was performed at the Clemson University Genome Institute. The DNA plugs were partially digested with EcoRI, and then DNA fragments size-fractioned (to ca. 120 kb) by pulse-field gel electrophoresis were ligated into pIndigoBAC536. Single colonies of Escherichia coli strain DH10b transformed with G. pectorale genomic fragment-containing BACs were picked, spotted on nylon membranes (or “BAC filters”), and stored frozen in glycerol at –80°C (total BAC number, plus: 27,648; minus: 18,432, estimated 24× and 16× coverage of G. pectorale plus and minus genome, respectively).