Primestar max dna polymerase premix

Degenerate primer pairs were designed based on relatively conserved regions of the mtDNA sequences of D. chinensis, D. dendriticum [19 (link)] and E. pancreaticum [20 (link)] and used to amplify the mitogenomes of three dicrocoelids (Additional file 2: Table S1). The complete mt genome of L. longicauda and the nearly complete mt genomes of Brachydistomum sp. and Brachylecithum sp. (excluding trnG, NCRs and trnE) were amplified in five or six overlapping fragments. Long PCR reactions were performed in reaction mixtures of 28 μl, containing 12.5 μl ddH₂O, 12.5 μl PrimeStar Max DNA polymerase premix (Takara, Dalian, China), 1 μl (10–40 ng) of template DNA and 1 μl (25 µM) of each primer. PCR cycling conditions were as follows: 98 °C for 2 min; 10 cycles of 92 °C for 10 s; 50–57 °C for 30 s; 68 °C for 1 min/kb followed by 92 °C for 2 min; 22 cycles of 92 °C for 10 s; 50–57 °C for 30 s; 68 °C for 1 min/kb and a final extension for 10 min at 68 °C. Positive amplicons were sequenced at Genewiz sequencing company.

The primers were designed based on relatively conserved regions of mtDNA sequences from Fasciola hepatica and Fasciola gigantica. The entire mt genome from a single specimen of F. magna was amplified in 5 overlapping fragments, using the primers shown in Additional file 1: Table S1.
PCR reactions were conducted in a total volume of 50 μl, using 25 μl PrimeStar Max DNA polymerase premix (Takara, Dalian, China), 25 pmol of each primer (synthesized in Genewiz, Suzhou, China), 0.5 μl DNA templates, and H₂O, in a thermocycler (Biometra, Göttingen, Germany). PCR cycling conditions started with an initial denaturation at 98 °C for 2 min, followed by 22 cycles of denaturation at 92 °C for 18 s, annealing at 52–65 °C for 12 s and extension at 60 °C for 1–5 min, followed by 92 °C denaturation for 2 min, plus 25 cycles of 92 °C for 18 s (denaturation), 50–67 °C for 12 s (annealing) and 66 °C for 3–6 min, with a final extension step for 10 min at 66 °C. A negative control (no DNA) was included in each amplification run. Amplicons (2.5 μl) were electrophoresed in a 2 % agarose gel, stained with Gold View I (Solarbio, Beijing, China) and photographed by GelDoc - It TS™ Imaging System (UVP, USA).

The ERG8 gene (NM_001182727.1) encoding the phosphomevalonate kinase in Saccharomyces cerevisiae was amplified by PCR containing PrimeSTAR MAX DNA polymerase premix (Takara Bio, Inc.) using the primers 5′-TCAGAGTTGAGAGCCTTCAGTGCCCCAG-3′ and 5′-GGAATTCTCTTTATCAAGATAAGTTTCCGGATCTTTTT-3′ and genomic DNA from S. cerevisiae as a template. pET21-d(+) was digested with NcoI, treated with the Klenow fragment of DNA polymerase I, and then digested with EcoRI. The PCR-generated DNA fragment was digested with EcoRI and ligated with the described vector fragment. The expected DNA sequence of the inserted fragment was confirmed by sequencing.
E. coli BL21(DE3) was transformed with the obtained plasmid and cultured in 20 ml of LB broth at 30 °C by reciprocal shaking at 140 r.p.m. When the OD₆₀₀ reached approximately 0.7, 0.1 mM IPTG was added and cultivation was continued overnight under the same conditions. Cells were harvested by centrifugation, resuspended in buffer solution A (50 mM sodium phosphate, 0.3 M NaCl and 20 mM imidazole) and disrupted by ultrasonication. After centrifugation, the resulting supernatant was adsorbed onto a His SpinTrap (GE Healthcare) column and the adsorbed proteins were eluted with eluting solution (buffer solution A containing 0.5 M imidazole). The obtained eluate was dialyzed with 20 mM Tris-HCl (pH 8.0) containing 50 mM NaCl as the external solution.