Takara taq hs

Selected density fractions of rRNA were reversely transcribed into complementary DNA (cDNA) using PrimeScript 1st strand cDNA synthesis kit (TaKaRa Biotech, Dalian, China) following the manufacturer's instructions, and subjected to pyrosequencing. The V4–V5 hypervariable regions of 16S rRNA genes were amplified using a primer set: 515f (5′-GTGCCAGCMGCCGCGG-3′) and 907r (5′-CCGTCAATTCMTTTRAGTTT-3′), containing the 454 FLX adapters and barcodes for sample identification (Zhou et al., 2011 (link)). Each 50-μl reaction mixture contained 2 μl of template cDNA, 0.8 μM of each primer, 0.4 mM of each dNTP (TaKaRa Bio, Otsu, Japan), 5 μl of 10 × PCR buffer (Mg²⁺ plus; TaKaRa Bio), 1.5 U of TaKaRa Taq HS and 10 μg of BSA (TaKaRa Bio). Amplifications were performed using the thermal conditions as reported earlier (Xu et al., 2014 (link)). After purification with Wizard sv gel and PCR clean-up system (Promega, Madison, WI, USA), equal amounts of the PCR products with different barcodes were mixed and submitted to the BGI Shenzhen (Shenzhen city, China) for pyrosequencing on a 454 GS FLX+ system.

Log in to GenBank and download the porcine NKL genomic sequence (GenBank Accession Number: NC_010445.4). The primers of all exons and partial adjacent introns of NKL gene were designed by Primers 5.0 software (Table 1). The primers were synthesized by Sangon Biotech (Shanghai, China).
DNA samples from 30 piglets were randomly selected to establish an equal amount of DNA mixed pool with 50 ng/L DNA concentration in each individual. The sample of DNA mixed pool was used as a template for PCR reaction. Here is the PCR reaction system (25 μL): 0.125 μL of TaKaRa Taq HS (5 U/μL; Takara Biotechnology, Dalian, China), 5 μL of 10 × PCR Buffer (Mg²⁺ plus), 2 μL of dNTP Mixture (2.5 mM), 50 ng of DNA samples, 10 pM of forward and reverse primers, ddH2O up to 25 μL. The following reaction conditions need to be followed correctly: denaturation at 94 °C for 5 min, then by 34 cycles at 94 °C for 30 s, 59–60.1 °C for 35 s, 72 °C for 35 s, and finally extend at 72 °C for 5 min. The qualified PCR products were sequenced by Sangon Biotech (Shanghai, China), and the sequencing results were analyzed, and SNPs detection were conducted by Chromes 2.3.1 and DNAMAN 6.0 software. According to the results of SNP identification, the SNPs from 300 piglets were genotyped by MALDI-TOF MS (Sequenom MassARRAY^®, Bioyong Technologies Inc., Beijing, China).