Hot start taq dna polymerase

Cryptosporidium species/genotypes were determined by nested PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene using primers F1 (5′-CCCATTTCCTTCGAAACAGGA-3′) and R1 (5′-TTCTAGAGCTAATACATGCG-3′) for primary amplification and F2 (5′-AAGGAGTAAGGAACAACCTCCA-3′) and R2 (5′-GGAAGGGTTGTATTTATTAGATAAAG-3′) for secondary amplification [16 , 17 (link)]. PCR reaction (25 μL) was composed of 1x PCR buffer (Mg²⁺-free), 2 mM MgCl₂, 200 μM of each deoxyribonucleoside triphosphate (dNTP), 0.4 μM of each primer, 0.2 U of HotStart Taq DNA polymerase (Takara, Dalian, China), and 2 μL of DNA template. The cycling conditions were 5 min at 95°C for initial denaturation and then 35 cycles of 45 s at 94°C, 45 s at 55°C, and 1 min at 72°C, followed by final extension at 72°C for 10 min. Both positive and negative controls were included in each test. PCR products were observed under UV light after electrophoresis in 1.5% agarose gel containing GoldView™ (Solarbio, Beijing, China).

Total RNA was isolated from HBE cells using the RNAiso Plus kit (Takara Biotechnology Co. Ltd., Dalian, China), according to the manufacturer's instructions. cDNA was synthesized from the RNA using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara Biotechnology Co. Ltd., Dalian, China). Real-time qPCR was performed using SYBR®Premix Ex Taq^TM II (Tli RNaseH Plus) (Takara Biotechnology Co. Ltd., Dalian, China) on an iCycler (ABI ViiATM7; Applied Biosystems, Carlsbad, CA, USA). The thermocycler parameters were set as follows: step one, activation of the HotStartTaq DNA polymerase (Takara Biotechnology Co. Ltd.) at 95°C for 30 sec; step two, PCR was performed for 40 cycles with denaturation at 95°C for 5 sec and annealing at 60°C for 34 sec; step three, fixed parameters set by the ABI 7500 Fast Real-time PCR system (Applied Biosystems)-associated SDS software version 2.3 (Applied Biosystems). Data were quantitated with 2^−ΔΔCt.
For each gene, an amplification curve was generated to evaluate the amplification efficiency. The sequences of the forward and reverse primers were: MFN2 forward 5'-CAGGTGTAAGGGACGATTGG-3' and reverse 5'-CAAATGGGATGAAGCACTGA-3'; GAPDH forward 5'-CAAATGGGATGAAGCACTGA-3' and reverse 5'-CGTCAAAGGTGGAGGAGTG-3'.

Total RNA was extracted using the RNAprep pure Plant Kit (Tiangen) with on-column DNaseI digestion from samples of 0, 1, 4 dpi. Reverse transcription was conducted with equal amount of total RNA (5 μg) using the SuperScript® III First-Strand Synthesis System (Invitrogen), and the resulting cDNA was diluted 1:3 with nuclease-free water.
Each PCR reaction used 3 μL cDNA. The PCR amplification was conducted using the Hot Start TaqDNA polymerase (Takara) in 50 μL volume. The following cycling condition was used: 95°C for 10 min and 38 cycles at 95°C for 15 s, 56°C for 30s and 72°C for 1 min. PCR products of IGYP genes in different infection time points were pooled separately, and 1 mL of pooling PCR products were gel-purified to remove >400-bp products and primer dimers and eluted by 200 μL ddH₂O.
The purified samples were used to build libraries using a TruSeq DNA sample prep kit (Illumina). The TruSeq libraries were sequenced using Illumina HiSeq2000 with 100-bp paired-end reads. Each library produced more than 2 Gb raw data. Low-quality bases (≦Q20), adaptors and short reads (≦50 bp) were removed. The sequences were aligned using TopHat2 software against the gDNA reference sequences of IGYPs[42 (link)].