M mulv rt buffer

RT-PCRs were performed in 2 steps. For the first cDNA synthesis, 100 ng total RNA, extracted from the latex of fruits or from plant tissues, was mixed with either 1.5 μM specific primers or an oligo-dT/random hexamer mix adjusted to 10 μL with ddH 2 O and incubated at 70°C for 5 min. Next, the RNA/primer mix was used in 20-μL reactions containing 10 U M-MuLV reverse transcriptase, 1X M-MuLV RT buffer, 0.25 mM dNTPs, and 0.25 mM dithiothreitol (all reagents from New England Biolabs). The reactions were incubated for 1 h at 42°C, 15 min at 70°C, and adjusted to a final volume of 50 μL. For second-strand synthesis, 50 μL PCRs contained 0.2 μM specific primers (Table 1), 0.2 mM dNTPs, 1.25 U Taq polymerase, 1X Thermo Pol buffer, and 2 μL first-strand cDNA. Reactions were performed using the following program: 94°C for 4 min, 30 cycles of 30 s for 94°C, 52°-60°C for 30 s, 72°C for 1 min, and a 10-min final extension at 72°C. Next, 5 μL was loaded onto a 0.8% agarose gel containing ethidium bromide. To assess the limit of detection (LOD), serial 10-fold dilutions of total RNA were used as a template in 25-μL 1-step RT-PCRs. The PCR products were purified using the QIAquick PCR purification kit (Qiagen) and ligated into the pGEMT-easy vector. Plasmid DNA from 3 positive clones was sequenced as described above.