Taq buffer

The potato endogenous beta-fructosidase gene (fru) and event-specific sequence of GM potato AV43-6-G7 were separately amplified using potato AV43-6-G7 DNA and 24 negative non potato AV43-6-G7 DNA samples.
Real-time PCR was performed with a real-time PCR instrument (Master Cycle Realplex4, Eppendorf, Germany). Each DNA sample was amplified in a 25-μL reaction in a 0.2-mL tube containing 200 nM each of primer F, primer R, and probe, 10× Taq Buffer (Transgene, Beijing, China), 2 mM magnesium chloride, 200 nM each of dATP, dCTP, dGTP and dTTP, 1.5 units of Taq DNA polymerase (Transgen, Beijing, China), and 100 ng of extracted DNA template [21 (link)]. After initial denaturation at 95 °C for 120 s, the PCR conditions were optimized through 45 cycles of amplification (at 95 °C for 15 s, and 60 °C for 30 s). In each RT-PCR setup, the reaction systems were filled with purified water (non template control, NTC) and negative sample DNA as negative controls. The same baseline was used for the threshold cycle (Ct) to compare the RT-PCR performances of the extracted DNA and negative DNA samples. For sensitivity tests of event-specific primers and probes, the concentration of the DNA template ranged from 0.001 to 100 ng.

Three plant DNA barcode loci (i.e., rbcL, ITS, and trnH‐psbA) were sequenced for each sample to increase the recovery of intact sequences from potentially highly degraded plant DNA from insect gut contents (Kress & Erickson, 2007; Kress et al., 2009; Li et al., 2011). The nucleotide sequences (5′ to 3′) of the primers are listed in Table S1. PCR was performed in 25 μl of solution containing 4 μl of DNA solution (10 ng/μl), 0.75 μl of each primer (10 μM), 2.5 μl of 10 × Taq buffer (TransGen Biotech), 0.5 μl of dNTP (2.5 mM), 0.25 μl of Easy Taq (5 units/μl) (TransGen Biotech), 0.75 μl of each primer (10 μM), and 16.25 μl of autoclaved distilled water. The PCRs were performed in Veriti 96‐well thermal cyclers (Applied Biosystems). The thermocycling program was as follows: 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 1 min, and a final extension of 72°C for 10 min. Amplified products (20 μl) were analyzed by electrophoresis on a 2% agarose gel in TAE buffer (40 mmol/L Tris‐acetate, 2 mmol/L Na₂EDTA·H₂O) and visualized with a UV transilluminator. Two positive [mungbean (Vigna radiata (L.) Wilczek) plant DNA] and two negative controls (PCR‐grade water instead of extracted insect DNA) were included in each PCR assay to determine amplification success and DNA carryover contamination, respectively.