Mangotaq dna polymerase

Two out of the 10 SNPs associated with AVG resistance were arbitrarily selected. PCR primers were designed to target the flanking regions of each of the two SNPs (Table 2). PCR was performed with three replicate samples of five cultivars representing the AVG resistant phenotype and 10 cultivars representing AVG susceptible phenotype (Table 1). Each PCR mixture contained 1 × MangoTaq reaction buffer (Bioline), 4 mM MgCl₂, 200 μM of dNTPs, 200 nM of each primer, 2% DMSO, 0.04 μg/μl BSA, 2 Units of MangoTaq DNA polymerase (Bioline), 2 μl of DNA template (≤10 ng/μl) and nuclease-free water to a final volume of 50 μl. Thermocycling conditions were an initial denaturation step at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, primer annealing at respective temperature (Table 2) for 30 s and extension at 72°C for 30 s, and a final extension step of 72°C for 5 min. PCR products were electrophoresed in a 1% agarose gel to confirm the presence of expected amplicons. Remaining PCR products were purified using a QIAGEN PCR purification kit according to the manufacturer’s instructions. Purified PCR products were sequenced at the Macrogen Inc, Seoul, South Korea, using the Sanger sequencing method (Sanger et al., 1977 (link)). Sequences were trimmed, processed, and aligned using Geneious R10.2.4 software to confirm the nucleotide base positions.

Parasitized DNA presence and integrity in 100 samples stored at -20°C (2007-2010) at FIDIC (from different areas of Colombia) were evaluated by 18S ribosomal RNA gene amplification using specific primers for P. vivax (SSU-F 5′-ATGAACGAGATCTTAACCTGC-3′ and SSU-R 5′-CATCACGATATGTA5TGATAAAGATTACC-3′) in a touchdown PCR [41 (link)]. The reaction contained: 1x Mango Taq reaction buffer (Bioline), 2.5 mM MgCl₂, 0.25 mM dNTPs, 0.5 mM of each primer, 0.1 U Mango Taq DNA polymerase (Bioline) and 10-40 ng gDNA in 10 mL final volume. The PCR thermal profile was: one initial denaturing cycle at 95°C (5 min), followed by ten cycles at 95°C (20 sec), annealing at 65°C (30 sec) and an extension step at 72°C (45 sec). Annealing temperature was reduced by 1°C in each cycle until reaching 55°C; 35 additional cycles were run at this temperature followed by a final extension cycle at 72°C (10 min). PCR products were visualized by electrophoresis on 1.5% agarose gel in 1× TAE, using 1 μL SYBR-Safe (Invitrogen).

Regenerated T₀ transgenic plants representing independent transformation events were identified by PCR using MangoTaq DNA polymerase (Bioline) with the primers listed in Supplementary Table S2 and genomic DNA as the template. T₀ and T₁ generation knockout plants were screened to determine whether a genomic cas9 gene was present, using N. tabacum GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (NtGAPDH) as a template control. Genome editing of NtCOL2a and NtCOL2b was analyzed by PCR using MyTaq DNA polymerase (Bioline) and the primers listed in Supplementary Table S2 for the amplification of exon I from genomic DNA. Starting with the T₀ generation, plants were screened by direct sequencing of purified amplicons. Because the transgenic T₀ plants were chimeras, selected individuals were analyzed in more detail by sequencing the amplicons following transfer to pCRII-TOPO (TOPO TA Cloning kit, Thermo Fisher Scientific).

Processed samples were used as template for PCR (10 μL final volume), containing 0.5 U/μL Mango Taq DNA polymerase (Bioline), 1× Mango Taq Color reaction Buffer, 2 mM MgCl₂, 250 nM dNTPs, 1 mM of each primer and DNase-free water to fulfil the necessary reaction volume. The PCR protocol for each fragment consisted of initial denaturing for 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 20 s at corresponding melting temperature and 30 s at 72°C. A reaction containing DNA-free water was used as negative control. The amplicons so obtained were purified with a Wizard PCR preps kit (Promega), once their quality has been evaluated on 3.25% agarose gel. A TOPO TA cloning kit was used for ligation, followed by transformation in TOP10 E. coli cells (Invitrogen). Several clones which grew on selective LB plates with 50 μg/mL kanamycin were incubated in LB broth at 37°C with 250 rpm overnight. Recombinant plasmids were purified using an UltraClean mini plasmid prep kit (MO BIO laboratories, California, USA) and sequenced with an automatic ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems, California, USA). Each insert’s integrity was checked by aligning the products with the respective theoretical sequenced fragments of each gene using Clustal W software [15 (link)].