Dna dilution buffer

Total RNA of MLO-Y4 with and without BDNF treatment for 24 h (n = 5) was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Then, the RNA was reverse transcribed with the Quantitect Kit (Qiagen), where first the contaminating DNA was removed by incubation with 2 µL DNA Wipeout buffer for 2 min at 42 °C. Afterwards, cDNA was diluted 1:4 with RNase-free water. For PCR, 4 µL diluted cDNA, 0.2 µL forward primer, 0.2 µL reverse primer (Table 1), and 0.6 µL RNase-free water were added to 5 µL Mastermix of the QuantiFast SYBR Green PCR Kit (Qiagen). The PCR was carried out in the LightCycler 2.0 (Roche) with the following conditions: 5 min denaturation at 95 °C, 40 cycles with 10 s denaturation at 95 °C and 30 s polymerization and elongation at 60 °C followed by melting curve analysis where the temperature increased stepwise from 60 to 95 °C. The melting curve proved the purity of PCR products. PCR products were additionally screened by gel electrophoresis using the QIAxcel Advanced System (Qiagen). Therefore, the samples were diluted 1:3 with DNA dilution buffer (Qiagen), attached to the QIAxcel Advanced System and compared with QX alignment marker (Qiagen). For semi-quantitative analyses, relative expression was calculated by the ΔΔ cycler threshold (Ct) method. βActin was used as reference gene.

We genotyped 20–24 worker offspring per mated queen that emerged seven weeks post oviposition initiation.
DNA was extracted using a Chelex protocol [62 (link)]. Five closely linked microsatellite loci (Table 1) were used to infer parental genotypes [63 ] using Mendelian inference. Multiplex PCRs were used to amplify 10 ng of DNA in 1 μl DNA dilution buffer (Qiagen), 400 pM of each primer, 1.25x reaction buffer (Sigma), 200 μM of each dNTP, 1U of Taq-polymerase and HPLC water to a final volume of 10 μl. The temperature profile for the PCR was as follows: 5 min denaturation at 95°C, 35 cycles of 30 sec each for denaturation (95°C), annealing T_m (Table 1) and extension (72°C), followed by a final step of 5 min at 72°C. The amplified products were separated in a MegaBace automated sequencer and fragment sizes were analyzed using the Fragment Profiler software. Alleles were scored as fragment lengths in base pairs.