Primestar gxl buffer

Two overlapping sets of primers were used to amplify the mitogenomes of the H. nepalensis and H. yeni species. The long-PCR primers were created using the 12S rRNA and cox1 genes of Haemaphysalis bancrofti (NC041076) and Haemaphysalis japonica (MG253031). The following PCR primers were employed:
The PCR was implemented in a 50 μl reaction mixture including 10 μl of 5X PrimeSTAR GXL Buffer (Takara, Japan), 1 μl of PrimeSTAR GXL DNA Polymerase (Takara, Japan), 4 μl of each primer, 4 μl of dNTPs, 4 μl of DNA template, and 23 μl of nuclease-free water. The PCR conditions used in the amplification procedure were as follows: initial denaturation at 95°C for 5 mins, followed by 45 cycles of denaturation (98°C for 10 s), annealing (68°C for 30 s), and extension (68°C for 10 mins), and a final extension was subjected to 68°C for 10 mins. The findings of the PCR were examined using 1.2 percent agarose gel electrophoresis stained with ethidium bromide (15 (link)). Libraries were sequenced on the Illumina HiSeq 2500 platform at Shanghai Biotechnology Co. Ltd. after the amplified PCR products had been purified.

PCR validation was performed using primers designed with PRIMER3 version 4.1.0 based on identified sequence variations (see S2 Table for primer sequences), such as deleted or newly introduced sequences and sequences flanking deletions. To generate PCR products up to 800 bp, each reaction contained: 10μl PCRBIO HS Taq Mix Red (PCRBiosystems), 7 μl ultrapure water (Biological Industries), 1 μl of each site-specific primer (10μM) and 1 μl of template genomic DNA (approximately 50 ng/μl). The PCR conditions were 95°C for 2 min, 35 cycles of 95°C for 10 sec, the calculated annealing temperature for 15 sec and 72°C for 15 sec. For PCR products longer than 800 bp, each reaction contained 12 μl ultrapure water (Biological Industries), 4 μl of 5X PrimeSTAR GXL Buffer (TaKaRa), 1.6 μl of 2.5 mM dNTPs, 0.5 μl of each site-specific primer (10 μM) and 0.4 μl of PrimeSTAR GXL DNA Polymerase (1.25 U, TaKaRa). The PCR conditions used were 94°C for 5 min, 30 cycles of 98°C for 10sec, the calculated annealing temperature for 15 sec and 68°C for 1 min. PCR products were visualized in 0.8–1% agarose gels. Note that expected PCR products were extracted from the agarose gel, and sequenced for validation. Figures were prepared using GIMP (https://www.gimp.org/), and Microsoft PowePoint.

Total RNA from ovules of flowers at stage 11 from two haploid-producing 4x T1 progeny (K6.1_20 and K6.1_23) of line RKD2gBB_6.1 was extracted, DNase treated and converted into single strand cDNA as previously stated. PCR reactions consisted of 2 µL of the first-strand cDNA synthesis reaction, 1× PrimeSTAR GXL Buffer, 200 µM dNTP, 0.2 µM primers, and 0.625 U PrimeSTAR GXL DNA Polymerase in a 25 µL reaction (Takara Bio USA, Inc) followed by 35 cycles of amplification with a cycling condition of 15 s at 98 °C, 15 s at 60 °C, and 2 min at 68 °C. Primers p1792/1801 (Table S1) were used to amplify the transcript from the start of the ORF through to the 3′ UTR. Amplified PCR products from each sample were directly cloned into PCR4-TOPO (Invitrogen) vector with cloned inserts sequenced at Psomagen (Rockville, MD, USA). Sequencing data were analyzed using Geneious prime software (Biomatters Limited, Auckland, New Zealand).