Taq pcr master mix kit

Total RNA was extracted from the peroneus longus of SD rats using the TRIzol reagent (Sangon Biotech Co., Ltd., Shanghai, China). One microliter of total RNA was used for reverse transcription reaction using the FastQuant RT Kit (Tiangen Biotech Co., Ltd., Beijing, China). Then, the 2x Taq PCR Master Mix kit (Tiangen Biotech Co., Ltd.) was used for PCR amplification. The PCR electrophoresis bands were photographed in the gel imager and the optical density values of the bands were measured using the Quantity One software (Bio-Rad Co., Ltd., California, USA). The optical density value of each objective gene band was divided by the optical density value of β-actin, which represents the mRNA expression level of the housekeeping gene. The sequences of GlyRS, MHC-IIb, and β-actin primers, which were designed and synthesized by Shanghai Sangon Biotech Co., Ltd., are as follows: 

GlyRS-F: 5′-GGTCAGTGTGAAGAGATTCCAG-3′.

 

GlyRS-R: 5′-AAGTCAATGGTGATGCCAAAC-3′.

 

MHC-IIb-F: 5′-GGCATTGAGTGGGAGTTCAT-3′.

 

MHC-IIb-R: 5′-GTCTTCAACCCGGACTTCTG-3′.

 

β-actin-F: 5′-GAGAGGGAAATCGTGCGTGAC-3′.

 

β-actin-R: 5′-CATCTGCTGGAAGGTGGACA-3′.

Male SD rates (average body weight 293.61 g) were purchased from the Experimental Animal Center of the Academy of Military Medical Science. All animal studies were approved by the Institutional Care and Use Committee of the hospital.
The 10% chloral hydrate, 70 U/ml heparin sodium, recombinant human serum albumin (rHSA), total superoxide dismutase (T-SOD) Kit, peroxidase (POD) Kit, blood urea nitrogen (BUN) Kit and total protein Kit was purchased from Nanjing Jiancheng Bioengineering Institute. The Quantscript RT Kit and 2XTaq PCR Master Mix Kit were obtained from Tiangen Biotech (Beijing) Co., Ltd.

cDNAs synthesized from mRNA of the adult melon aphid using a 2×Taq PCR Master Mix kit (TIANGEN, KT201-02) were used to amplify a linear template for dsRNA synthesis (refer primer sequences of aphids in Table S4). The amplified sequences were introduced into the pMD19T-Vector (Takara, Kyoto, Japan, 3271) and transformed into DH5α competent cells (Invitrogen, Carlsbad, CA, USA, BC102-02). The plasmids were extracted and verified by Sanger sequencing, then used as the template for dsRNA synthesis, using a T7 RiboMAX Express RNAi System (Promega, Madison, WI, USA, P1700). The T7 promoter sequence was incorporated with the aphid primer sequences for dsRNA synthesis (Table S4) (Tsingke Biotechnology, Beijing, China). The EGFP-dsRNA, which targets the exogenous gene enhanced green fluorescent protein (EGFP), was used as a nonspecific control.

Two pairs of primers were designed for the almA and alkB genes (almA-F, 5′-CGTGACAACGAAGACTGCATTAC-3′; almA-R, 5′-CGTGACAACGAAGACTGCATTAC-3′; alkB-F, 5′-TTGCGTTTGAAAAAGTGGGG-3′; alkB-R, 5′-CGGTAGCTCTTGTCCTGGGA-3′), based on the gene predictions for the genome. PCR amplification of the genes was performed with the 2× Taq PCR Master Mix kit (TianGen, Dalian, China). The fragments were cloned into the pMD18-T vector (TaKaRa) and then transformed into competent JM109 cells (TaKaRa). Colony PCR was performed by direct PCR with the M13F and M13R primers. After bacterial culture, the plasmid was extracted using a plasmid midi kit (Omega, Norcross, GA, USA). The DNA was serially diluted 10-fold, as were the standard PCR products. The gene copy numbers were estimated by qPCR in triplicate, using the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA) with SYBR green I (TaKaRa). The qPCR was performed in a 20-μl reaction mixture with 2 μl of template DNA (1 ng), 0.8 μl of each primer (10 nM), 0.4 μl of ROX reference dye, and 6 μl of sterile water. The thermal cycling conditions were 95°C for 30 s, 40 cycles of 95°C for 5 s and 60°C for 30 s, and a melting curve stage of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s.