Using a two-fold serial dilution that ranged from 100 ng to 3.125 ng, the DNA for each concentration was digested in triplicate with HpaII, MspI and treated with buffer only.
The PCB concentrates was digested with aqua regia, cooled, and then diluted to appropriate concentration range using deionized water.
For our own protocol, 200 μl lysis buffer (4 M guanidinium thiocyanate, 30 mM Tris, pH 8.0, 1% Triton-X-100) was added to the dried sections and immediately homogenized in a Mixer Mill at 20 Hz for 4 min. Proteinase K (Roche Diagnostics, Mannheim, Germany) was added (1 mg/ml final concentration) and tissue was digested for 1 hour at 55°C.
The gene mixture at a total concentration of 23 ng/µl was digested by DNaseI (0.001 U/µl) in a buffer of 10 mM MgCl2, 50 mM Tris, pH 7.4 at 20°C for 2.5'.
Naked DNA was digested with multiple concentrations of DNase I to rule out sequence-based bias of DNase I digestion [ 4].
To determine trace element concentrations, 50 mg of feather powder was digested in 1 ml of nitric acid (69 70%) and 0.5 ml of hydrogen peroxide (30%) using Teflon® bombs during 12 hours at 60°C.
As shown in Figure 7B, the N-terminal portion of LiTXN3 (detected with the anti-His antibody) was digested at digitonin concentrations that expose cytosolic proteins to PK (at 0.8 mg digitonin/mg protein; controlled with the anti-LicTXNPx2 antibody), but that do not enable PK to reach the mitochondrial compartments.
Chymotrypsin cleavage: Procollagen was digested with variable concentrations of chymotrypsin (Sigma, C7762) in 1X storage buffer, in volumes of 20 μl for 30 min at 4 °C.
The pretreated orange peel waste was digested at different concentration of volatile solids ranging from 0.5 to 2%.
Approximately 250 300 mg of the ground powder was digested and the metal concentration was measured.
