Amplification of invertase was performed by 50 cycles of incubation at 94 °C for 30 s followed by a 45-s incubation at 63.2 °C. After the initial denaturation, amplification of cruciferin was performed at 40 cycles of incubation at 95 °C for 30 s, 59 °C for 1 min and 72 °C for 30 s. Finally, for the qPCR amplification of 16S rDNA, the initial denaturation at 95 °C for 5 min was followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C
for 30 s and elongation at 72 °C for 30 s. In the particular case of 16S rDNA amplification, a final elongation at 72 °C for 10 min was also included. In all cases, melting curve analysis was performed at a temperature range of 65–95.1 °C. Qualitative and quantitative analysis of the DNA obtained during the optimization of the DNA extraction method was performed by UV spectrophotometry
(Table 2; Supporting Information, Tables S1–S4). Obeticholic Acid in vivo Although starting quantities of rumen fluid and plant material differ because of limitations associated with the see more different techniques, clearly the yields of DNA obtained were extremely variable, ranging from undetectable to 800 ng μL−1. Generally, the highest yields combined with the optimal A260 nm/A280 nm ratios were obtained using CTAB. The statistical significance of the data obtained by this method was analysed by anova (Table 3). This analysis demonstrated that the reagents used for the extraction had significant effects on the yield of DNA extracted from rapeseed and maize. Namely, the yield was significantly higher when DNA was extracted twice with phenol : chloroform : isoamyl alcohol, than when phenol was omitted from the extraction reagent. Inclusion of phenol in the extraction buffer did not yield higher amounts of DNA for soya, but the quality of DNA was significantly higher when the extraction reagent included Ribose-5-phosphate isomerase phenol. The amount of starting material used for each extraction did not have any significant effects for rapeseed. On the other hand, 50 mg of starting material appeared to be the optimum for DNA extracted from maize. In the particular case of soya, the amount of extracted
DNA appeared to be directly correlated with the amount of starting material used for the extraction (Table 3). Agarose gel electrophoresis verified the results obtained by UV spectrophotometry. Thus, exclusion of phenol from the extraction buffer resulted in the presence of contaminating substances in soya and rapeseed DNA that were retained in the wells (Fig. 1). As these substances did not appear to have any significant effects on the A260 nm/A280 nm ratio obtained by the Nanodrop, it was assumed that the co-precipitating substances were humic acids. Humic acids absorb UV light at a similar wavelength to that of nucleic acids (254 nm), thus would not affect the A260 nm/A280 nm ratio, but they are unable to penetrate agarose gels.