Pcr buffer

Genomic DNA was extracted from 1–2 legs (dry or in 100% ethanol) of each specimen (Table 1) using an animal tissue DNA extraction kit (Huier nanotechnology, Luoyang, China) according to the manufacturer’s protocol. The DNA was dissolved in a 100 μl elution buffer and stored at -20°C. Partial COI genes were amplified by PCR with the universal primers Ron (5’-GGATCACCTGATATAGCATTCCC-3’) and Nancy (5’-CCCGGTAAAATTAAAATATAAACTTC-3’) [6 (link)]. For each PCR, a 20 μl mixture containing 2 μl template DNA, 2 μl 10×PCR buffer (Sangon biotech, Shanghai, China), 0.3 U Taq polymerase (Sangon biotech, Shanghai, China), 2.5 mM MgCl₂, 0.5 μl dNTPs (10 mM each) and 0.5 μl (10 μM) forward and reverse primers was used under the reaction conditions described by Hebert et al. [7 (link)] on a thermal cycler (AB, the USA). The ~500bp PCR products were sequenced by Sangon biotech, Shanghai, China. DNA sequences were first aligned in Clustal X 2.0 software [8 (link)], and then a neighbor-joining (NJ) tree based on the Kimura-2-parameter distance model [9 (link)] was generated by MEGA6 software [10 (link)] to quantify sequence divergences among specimens. Nodal support of the NJ tree was estimated by 1000 bootstraps. All sequences obtained in this paper were deposited in GenBank (Table 1).

Total RNA was extracted from mango leaves using the RNAprep Pure Plant Kit (TianGen, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg of total RNA using M-MLV reverse transcriptase (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Genomic DNA was extracted from mango leaves using the CTAB method. Four TFL1 genes were obtained from mango leaves and named MiTFL1-1, MiTFL1-2, MiTFL1-3 and MiTFL1-4. Specific primers (QTFL1-1u/d, QTFL1-2u/d, QTFL1-3u/d, and QTFL1-4u/d; Table S2) were designed to amplify MiTFL1 genes from genomic DNA and cDNA. The polymerase chain reaction (PCR) mixture contained 2.5 μl of 10× PCR buffer (with MgCl²⁺), 0.5 μl of 10 mM dNTPs (Sangon Biotech, Shanghai, China), 1 μl of each of the upstream and downstream primers (10 μM), 0.15 μl of TransTaq-T DNA polymerase (TianGen), 1 μl of genomic DNA (100 ng/μl) or cDNA (100 ng/μl), which served as the templates, and sterile water (25 μl). The PCR amplification conditions included an initial denaturation step of 4 min at 95 °C; 38 cycles of 95 °C for 40 s, 56 °C for 50 s, and 72 °C for N min (N = 1 min/kb); and a final extension at 72 °C for 10 min. The amplified fragments were cloned into the pMD18-T vector (Takara) and then sequenced.