, 2006, Community Reference Laboratory, (CRLV04/05XP)]. In these instances, the recommended amount of starting material was used for each extraction. Three protocols for DNA extraction using PARP phosphorylation the CTAB-based DNA extraction method are described, with the method of choice dependent on the extraction scale required. The CTAB lysis buffer contained 2% w/v CTAB (Sigma-Aldrich, Poole, UK), 100 mM Tris–HCl (pH = 8.0; Fisher), 20 mM EDTA (pH = 8.0; Fisher) and 1.4 M NaCl (Fisher).

The pH of the lysis buffer was adjusted to 5.0 prior to sterilization by autoclaving (Doyle & Doyle, 1987). Standard method in 2.0-mL microcentrifuge tubes: The original samples used in all the protocols described herein consisted of 1.8 mL of rumen fluid and 50 mg of ground plant seed material. Samples were lyophilized at − 40 °C for 48 h and bead-beaten on a prechilled rack at − 80 °C for 1 min using 8-mm glass beads (Fisher). For the optimized protocol, 50 mg of lyophilized material

was thoroughly mixed with 900 μL of CTAB lysis buffer. All samples were incubated at 65 °C for 60 min before being centrifuged at 12 000 g see more for 5 min at 4 °C. Supernatants were transferred to fresh 2-mL microcentrifuge tubes and 900 μL of phenol: chloroform: isoamyl alcohol (25 : 24 : 1, pH = 6.7; Sigma-Aldrich) added for each extraction. Samples were mixed thoroughly prior to being incubated at room temperature for 10 min. Phase separation occurred by centrifugation at 12 000 g for 15 min at 4 °C, and the upper aqueous phase was re-extracted with a further

900 μL of phenol:chloroform:isoamyl alcohol. Next, samples were centrifuged at 12 000 g for 10 min at 4 °C, and the upper aqueous phases were transferred to fresh 2-mL microcentrifuge tubes. The final extraction was performed with 900 μL of chloroform: isoamyl alcohol (24 : 1), and layer separation occurred by centrifugation at 12 000 g for 15 min at 4 °C. Precipitation of DNA was achieved by adding the upper phase from the last extraction step to 450 μL of isopropanol (Sigma-Aldrich) containing 50 μL of 7.5 M ammonium acetate (Fisher). Samples were incubated at −20 °C overnight, Orotidine 5′-phosphate decarboxylase although shorter incubations (1 h) produced lower DNA yields. Samples were centrifuged at 7500 g for 10 min at 4 °C, and supernatants were discarded. Finally, DNA pellets were washed three times in 1 mL of 70% (v/v) ethanol (Fisher). The final pellet was air-dried and re-suspended in 200 μL of 75 mM TE buffer (pH = 8.0; Sigma-Aldrich). High-Throughput (96-well plate format): Twenty millligrams of starting material was used for each DNA extraction in the high-throughput format. 110 μL of CTAB lysis buffer was added to each sample, and samples were incubated at 65 °C for 60 min. Samples were extracted twice with 110 μL of phenol: chloroform: isoamyl alcohol (25 : 24 : 1, pH = 6.7; Sigma-Aldrich) and once with 110 μL of chloroform: isoamyl alcohol (24 : 1; Sigma-Aldrich).