Hindiii restriction endonuclease

Restriction endonuclease treatment of the genomic samples was used to evaluate the side effects of the pretreatments on the final result. A 50 µL reaction volume was prepared, containing 5 µL of 10× buffer 2.1 (New England Biolabs, Beijing, China), 20 U of HindIII restriction endonuclease (New England Biolabs, Beijing, China), 2500 ng of the genome sample and ddH₂O up to 50 µL. The reaction was performed at 37 °C for 2 h. Following digestion, the DNA was isolated using a QIAquick PCR Purification Kit (Qiagen, Duesseldorf, Germany).

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.

Genomic DNA was isolated from 300 to 400 mg of rice leaf tissue that had been ground in liquid nitrogen using 500 μl CTAB extraction buffer (1.5% CTAB, 1.05 M NaCl, 75 mM Tris-HCl, 15 mM EDTA pH 8.0) [31 (link)]. After incubating at 65 °C for 1 h, samples were thoroughly mixed with equal volumes of chloroform:isoamylalcohol (24:1) and centrifuged at 13000 rpm for 10 min. The aqueous layer was transferred to fresh tubes, precipitated by mixing with equal volumes of isopropanol and centrifuged for 10–15 min at 13000 rpm. Pellets were washed with 70% ethanol, air-dried and dissolved in 50 μl dd H₂O.
For each transgenic plant, 10 μg genomic DNA was digested with the HindIII restriction endonuclease (New England Biolabs). Following electrophoresis in a 1% agarose gel stained with SYBR Safe (Invitrogen), digested DNA was transferred onto Hybond N+ membrane (GE Healthcare, UK). Blots were hybridized with a digoxygenin (DIG)-labelled specific DNA probe for the HYG gene and signals were detected using CDP-Star according to the manufacturer’s instructions (Roche Diagnostics).