Primestar hs dna polymerase kit

Total RNA was extracted from chrysanthemum flowers using the MiniBEST Plant RNA Extraction Kit (TaKaRa). First-strand cDNA was synthesized using a reverse transcription system (Promega). Primers specific for the TM6 ORF (Table 5) were designed on the basis of a sequence annotated as TM6 in the transcriptome data for chrysanthemum flowers. The PrimeSTAR HS DNA polymerase kit (TaKaRa) was used for amplifying the TM6 ORF. The PCR product was examined by 1% agarose gel electrophoresis. The amplified fragment purified from the agarose gel was inserted into the pGEM-T Easy vector (Promega) and sequenced by the Shanghai Sangon Company.

Microbial DNA was extracted from 250 mg of each fecal sample (n = 906) using the commercial FastDNA^™ SPIN Kit for Soil (MP Biomedical, Solon, OH, United States). DNA was quantified with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) and stored at −20°C. Amplicon sequencing libraries preparation of the V1-V2 region of the 16S rRNA gene was carried out with PrimeSTAR^® HS DNA Polymerase kit (TaKaRa, Beijing, China) following the method described in Kaewtapee et al. (2017) (link). Briefly, 1 μL of DNA was added for the first PCR, in a 20-μl reaction with 0.2 μL of PrimeSTAR HS DNA polymerase and 0.5 μL of each primer. The second PCR, which used 1 μL of the first PCR as a DNA template, ran in a total volume of 50 μL. An initial denaturation at 95°C for 3 min was followed by 15 cycles (first PCR) or 20 cycles (second PCR) of denaturation at 98°C for 10 s, subsequent annealing at 55°C for 10 s, extension step at 72°C for 45 s and a final extension for 2 min at 72°C. Derived amplicons were inspected by agarose gel electrophoresis, purified, and normalized using SequalPrep Normalization Kit (Invitrogen Inc., Carlsbad, CA, United States). Samples were sequenced using 250 base pairs (bp) paired-end sequencing chemistry on an Illumina Novaseq 6,000 platform.

RNA isolated from Taxus yunnanensis Cheng et L. K. Fu twigs was converted into cDNA using the PrimeScript RT reagent Kit with gDNA eraser (RR047A, Takara). In general, the open reading frames (ORFs) of candidate genes were cloned from Taxus twig cDNA with PrimeSTAR HS DNA Polymerase kit (R010A, Takara). Then PCR products were linked into the pMD18T vector (D101A, Takara) and transformed into E. coli TOP10 cells. Transformants were plated for selection on LB plates with 100 μg/mL ampicillin and positive colonies were verified by colony PCR and Sanger sequencing.

Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco′s modified Eagle′s medium (DMEM) with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 250 ng/mL amphotericin B (Invitrogen) and incubated at 5% CO₂ at 37 °C. 400–600 bp wild type DNA fragments of the 3′UTRs of fas, lxrα, ddit3, casp3a, baxa and lamp2 mRNA containing the putative binding sites of miR-205 were synthesized by PCR. The fragments were then subcloned into the XhoI and NotI sites downstream of the Renilla luciferase coding region in the psiCHECK-2 vector (Promega, Madison, WI, USA), and ligated using ClonExpress™ II One Step Cloning Kit (Vazyme, Piscataway, NJ, USA). Mutant recombinant plasmid was only mutates 6mer which was complementary to miR-205 seed sequence to GACCTA by overlap-PCR, validated by sequencing by Tsingke (Wuhan, China). The PCR reactions were performed using TaKaRa PrimeSTAR^® HS DNA Polymerase kit as mentioned above. HEK-293T cells were plated at 1 × 10⁵ cells/well on 24-well plates and co-transfected with psiCHECK2-fas-3′UTR (psi-fas) (0.2 μg), psiCHECK2-mutfas-3′UTR (psi-fas-mut) (0.2 μg), respectively, or empty vector plasmid (0.2 μg) and miR-205 mimics (20 pmol), miR-205 negative control (20 pmol) using Lipofectamine2000 (Invitrogen) (2 μL). Cells were harvested at 48h transfection.