Ex taq pcr kit

Both RT-PCR and qRT-PCR were performed as previously reported⁴⁵ . Briefly, total RNA was extracted using the RNeasy Kit (Qiagen) according to the manufacturer’s instruction and quantified by Nanodrop 8000 (Thermo Fisher Scientific). cDNA was obtained by reverse transcription using ReverTra Ace (Toyobo). PCR was performed with the Ex-Taq PCR Kit (Takara Bio) according to the manufacturer’s protocol using a thermal cycler (Veriti96-Well Thermal Cycler; Thermo Fisher Scientific). The PCR cycles were as follows. For β-ACTIN and GAPDH, initial denaturation at 94 °C for 2.5 min, followed by 25 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min. For the other genes, the cycles consisted of an initial denaturation at 94 °C for 2.5 min, followed by 35–40 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min. qRT-PCR was performed using SYBR Green PCR Master Mix (Takara Bio) and the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Denaturation was performed at 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and at 60 °C for 30 s. The threshold cycle method was used to analyze the data for gene expression levels, and the values were calibrated to those of the housekeeping gene β-ACTIN or GAPDH. The primer sequences used in this study are listed in supplementary Table S2.

The extracted total RNA was reverse-transcripted into cDNA using PrimeScript RT reagent Kit (TAKARA cat#RR047A) according to the manufacturer's protocol in triplicates. The resulting cDNA was pooled for next PCR amplification. Primer sequences are shown in Supplementary Table 1. The target sequences were amplified by PCR in 50μl of 1×Taq buffer containing 0.3μM of each primer, 1.5 mM magnesium chloride, 200μM dNTP mixture, 2.5 units of Taq polymerase and 1μl (10μg) of each cDNA by Ex Taq PCR kit (TAKARA cat#RR001A). The reaction was started after 5min denaturation of cDNA at 94°C (hot start). DNA amplification in a T100 cycler (Bio-Rad Laboratories, Inc) was followed by a final extension for 8min at 72°C for HJURP, ADAMTS8 and GPT2 (94°C 30s, 58.5°C 1min; 40 cycles). Although a range of annealing temperatures (from 5°C below the Tm to 5°C above the Tm) had been tried, the PCR products of other 9 mRNAs (TOP2A, GINS2, TK1, CDCA5, AGER, FHL1 CLDN18, ADH1B, and GPIHBP1) were rare compared with positive control. GAPDH expression was used as an internal control. All PCR products were visualized by 2% agarose gels electrophoresis. Water negative controls contained all components for the RT-PCR reaction without target RNA. Positive controls of RNA were extracted from A594 cells obtained from the Cancer Center of Xiamen University (Xiamen, China).

The total RNA of each sample was isolated with TRIzol™ Reagent (Invitrogen) according to the manufacturer’s instructions. The first cDNAs were synthesized using the ExTaq™ PCR Kit following the manufacturer’s protocol (Takara, China). qRT-PCR was carried out with a BIORAD CF96 Real-Time PCR system using SYBR ^® Premix Ex Taq™ II (Takara, China). The primers used for qRT-PCR are listed in Table S4, and the β-tubulin gene was used as an internal reference. Each treatment was repeated three times independently. The 2^−∆∆Ct method was used to analyse the relative expression of genes.

Total RNA was isolated using the RNA Extraction Kit (Takara, product no. 9767) in accordance with the manufacturer's recommended protocol. The first-strand cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, product no. RR047A) for RT-PCR. In brief, a 10 μl system containing 2 μl of 5 × PrimeScript RT Master Mix (Perfect Real Time) and RNase-Free Distilled Water and RNA solution, with a total RNA amount of 500 ng, was transferred to the ABI ProFlex PCR System (2 × 96-well) for reverse transcription. RT-PCR was then conducted using the Ex-Taq PCR kit (Takara, product no. RR820A) following the manufacturer's instructions. All RT-qPCR primer sequences were included in Additional file 2: Table S1.

Linear DNA fragments were amplified using the ExTaq PCR kit (Takara) and the appropriate plasmid template. Standard 25 μl translation reactions were set up containing 0.5 μg linear DNA, 10 μl premix (Lesley et al. (1991) [20 (link)]), 2.5 μl 1 mM each l-amino acid (except methionine), 7.5 μl S-30 extract, 10 μCi [³⁵S] methionine, 1 μl of 5 μg/μl anti-ssrA oligonucleotide (5′-TTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCGACTA-3′). Reactions were incubated at 37°C for 30 min, then chilled on ice for 5 min and then mixed with 10 volumes of 2% (w/v) Cetyltrimethylammonium bromide (CTABr) and 10 volumes of 0.5M NaOAc (pH 4.7) and incubated on ice for a further 15 min before being centrifuged at room temperature (13400 rpm, 10 min). CTABr pellets were washed with 500 μl cold acetone. CTABr supernatant was incubated with 10% TCA on ice for 10 min before being centrifuged at 4°C (14000 rpm, 10 min) and TCA pellets were then washed with 1 ml cold acetone. All pellets were then centrifuged at 4°C (14000 rpm, 10 min). All samples were re-suspended in sample buffer and analysed by SDS/PAGE.

The Pre. intermedia/nigrescens group-specific repeat (PINSR)-specific primers PINSR-F and PINSR-R (Supplementary Table S2) were designed from the consensus sequence of PINSR. The expected product length was 149 bp. PCR amplification was performed with 50 ng of genomic DNA and an Ex Taq PCR kit (Takara Bio) using the following temperature program: 94°C for 1 min and 30 cycles of 94°C for 20 s, 51°C for 30 s, and 72°C for 30 s. The amplified products were detected by agarose gel electrophoresis followed by ethidium bromide straining. As a control, the 16S-rRNA gene was amplified using the primers 16S-rRNA-10F and 16S-rRNA-800 (Supplementary Table S2).