Gotaq pcr master mix

Total RNA was extracted from rice tissues using an RNA Extraction Kit (Macrogen, South Korea) according to manufacturer instructions. First-strand cDNA was synthesized with 2 µg of total RNA in a 25 µl volume using M-MLV reverse transcriptase and oligo(dT)₁₅ primer (Promega), and diluted with 75 µl of water. The qPCR amplifications were conducted on the LightCycler 2.0 instrument (Roche Diagnostics). The 20 µl of qPCR mixture included 2 µl of the first-strand cDNA mixture, 10 µl of 2X GoTaq PCR Master Mix (Promega), and 1 µl of 10 pM primer pairs (Supplementary Table S1). The qPCR conditions were 95°C for 2 min, followed by 50 cycles at 95°C for 5 s, 59°C for 15 s, and 72°C for 10 s. The rice UBIQUITIN5 (OsUBQ5, AK061988) gene was used as an internal control for normalization. The semi-quantitative PCR was performed in a 20 µl volume containing 2 µl diluted cDNA, 1 unit Ex Taq polymerase (TaKaRa Biotechniques), and 1 µl of 10 pM primers (listed in Supplementary Table S1). The PCR program included initial denaturation at 94°C for 3 min, followed by specified cycles at 94°C for 30 sec, 55°C for 1 min, and 72°C for 40 sec, followed by a final extension at 72°C for 5 min. The PCR products were electrophoresed on a 1% agarose gel. OsUBQ5 was used as an equal loading control.

DNA constructs were chemically synthesized (gBlocks dsDNA fragments, IDT) with M13F and M13R homologous regions at each end. To generate a dual biotin-labeled DNA substrate, PCR reactions were performed using a 2x GoTaq PCR master mix (Promega), biotin-labeled M13F and biotin-labeled M13R primers, and gBlocks fragments as templates. PCR products were gel purified, and subsequently used in BLI assays.

PCR amplification of lung SOD and CAT was performed using specific pair of primers (synthesized by Macrogen Co., GAsa-dong, Geumcheon-gu., Korea) for rat. The sequences of specific primers and product sizes are listed in Table 2.
4 microliters of cDNA and 1.25 μM of each primer were added to 12.5μM 2xGoTaq PCR master mix (Promega Corporation, Madison, WI, USA). The volume was completed to 25 μl with DNase free water and samples were loaded into Techne TC-3000x thermal Cycler (Bibby Scientific, England). PCR conditions were 94 C for 5 mins followed by cycles of 1 min at 95°C, 1 min at annealing temperature 60°C and 1 min at 72°C. The cycle number was adjusted so that all reactions were within the linear range of product amplification according to the reference gene (28 cycles). The final extension step was 7 min at 72°C, PCR products were separated on 1.5% agarose-A (Bio Basic, Markham, ON, Canada) gel in 1.0 X-TAE (Tris–Acetate-EDTA) buffer (Sigma–Aldrich, St. Louis, MO, USA) at 100 V for 30 min.
The gel was stained with ethidium bromide (Sigma–Aldrich), visualized and photographed under UV light using Ingenius gel documentation system (Syngene Europe, Cambridge, UK) [20 ].