Taq pcr reagent kit

Laboratory common reagents, such as ethanol, sucrose, paraformaldehyde, hematoxylin, eosin, agarose and electrophoresis reagents, were purchased from Beijing Bioland Technology Company. The Universal DNA Purification kit was a product of the TIANGEN Biotech Company, China. The DNeasy Plant Mini Kit was a product of Qiagen Company, Germany. The Taq PCR reagent kit and Vector NTI Advance 9 software were purchased from Invitrogen, United States.
Reagents and biochips required for MassARRAY SNP MALDI-TOF MS genotyping were from Sequenom, USA (acquired by Agena Bioscience Company). The SNP MALDI-TOF MS genotyping experiments were performed at CapitalBio Co., Beijing, using a 384-well biochip (SpectroCHIP, Sequenom) and a commercial “iLEX Gold Cocktail” (Sequenom; exact formula unavailable) containing all necessary PCR reagents, including polymerase and dNTPs, and dideoxy A, T, C, and G terminator nucleotides (ddNTPs) to control DNA chain elongation.

Laboratory common reagents such as ethanol, sucrose, paraformaldehyde, hematoxylin, eosin, agarose and electrophoresis reagents, etc. were purchased from Beijing Bioland Technology Company. Mercuric chloride (0.1%) for surface sterilization of freshly collected C. sinensis specimens was a gift from the Institute of Microbiology, Chinese Academy of Sciences. EcoRI endonuclease is a product of New England BioLabs, United States. The Universal DNA Purification kit was a product of TIANGEN BIOTECH Company, China. The DNeasy Plant Mini Kit was a product of Qiagen Company, Germany. The Gel Extraction Kit was a product of Omega Bio-Tek, United States. The Taq PCR reagent kit and Vector NTI Advance 9 software were purchased from Invitrogen, United States. Calmodulin was a product of Abicom (Shanghai, China).

The genomic DNA templates and aforementioned universal, genotype- and species-specific primers were used in PCR assays using a PCR instrument (Bio-Rad, United States) and Taq PCR reagent kit (Invitrogen) to amplify the ITS1-5.8S-ITS2 segments using the following touch-down protocol: (1) 95°C for 5 min; (2) 36 cycles of 95°C for 30 sec, annealing temperature for 30 sec (the annealing temperature was initially set to 70°C and decreased stepwise by 0.3°C in each cycle), and 72°C for 1 min; (3) 72°C for 10 min and final incubation at 4°C [26 , 27 ]. The PCR amplicons were examined by agarose gel electrophoresis and sequencing.