Uv transilluminator

RILs and parental genomic DNA was extracted from the leaves of 21 days old seedlings by following CTAB method of Murray and Thompson [46 (link)]. PCR was carried out in a total volume of 15μl, the ingredients include double distilled water(ddH₂O), 10X buffer (100mM Tris-HCl with pH 8.8; 500mM KCl; 1% Triton X-100; 16mM MgCl₂), dNTPs, primer, Taq polymerase (Bangalore Genei, India) and DNA. The proportion of ingredients for 15μl PCR mixture for Xbarc primer series: ddH₂O(10.2μl), 10X buffer(1.0μl), DNA(2.0μl), dNTPs(0.5μl), forward and reverse primer(1.0μl) each and Taq polymerase(0.3μl); for Xwmc primer series: ddH₂O(8.4μl), 10X buffer(1.5μl), DNA(2.0 μl), dNTPs(0.6μl), forward and reverse primer(1.0μl) each and Taq polymerase(0.3μl); for Xcfa, Xcfb, Xcfd and Xgdm primer series: ddH₂O(9.1μl), 10X buffer(1.5 μl), DNA(2.0μl), dNTPs(0.6μl), forward and reverse primer(1.0μl) each and Taq polymerase(0.25μl); for Xgwm primer series: ddH₂O(10.2μl), 10X buffer(1.0μl), DNA(2.0μl), dNTPs(0.5μl), forward and reverse primer(0.5μl) each and Taq polymerase (0.3μl). The amplified PCR products were resolved in 3.5% agarose at 120V for 3 hours in TBE buffer. Wherever the resolution was low, the amplified PCR products were separated by electrophoresis on 4% metaphor agarose. PCR amplified products were visualized and photographed on a UV transilluminator (Syngene) for further scoring.

DNA molecular analysis was carried out using published PCR-relevant protocols [15 (link), 19 (link)]. In brief, the genotyping analysis was performed using assemblages-specific primers that amplify, in the case of the PCR-tpi, a 148-bp fragment of the assemblage A and 81-bp fragment of assemblage B [19 (link)]. The other PCR (PCR-E1-HP) amplifies a 165-bp amplicon for assemblage A and 272-bp fragment for assemblage B [15 (link)].
The PCR reaction mixture was done using a AmpliTaq DNA Polymerase with GeneAmp 10x PCR buffer Kit (Applied Biosystems, USA) in a total volume of 25 μL and comprised 10 μL of 10x PCR buffer (Applied Biosystems, USA), 0.2 mM of each deoxynucleoside triphosphate (dNTP) (Applied Biosystems, USA), 1 U of Taq polymerase (Applied Biosystems, USA), 0.4 μM of each primer, and 5 μL of DNA template, with ultrapure water used as a negative control.
The DNA was amplified using a thermocycler (Gene Amp PCR System 9700, Applied Biosystems, USA). The PCR products were analyzed by 2% agarose gel electrophoresis, stained with 0.5 μg/mL of ethidium bromide, and then visualized on a UV transilluminator (Syngene, U:Genius, Belgium).
DNA from axenic cultures of G. duodenalis strains WB-C6 (assemblage A) and GS (assemblage B) was used as positive controls, while ultrapure water was included in negative controls.

A random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR) was used for characterization of the V. vulnificus isolated isolates. The RAPD11 primer used was 5′-AAAGCTGCGG-3′ and RAPD15 5′-CACACTCCAG-3′. The PCR technique was carried out in 0.2-μL microfuge tubes. The total volume of the reaction mixture was 50 μL, consisting of 25 μL 10× PCR master mix (EconoTaq^®PULS GREEN 2X Master Mix, Lucigen, Middleton, WI, USA), 0.5 μL of OPC primer, and 1.0 μL (10–20 ng) of template DNA, the volume was then adjusted to a final volume by adding Nuclease Free Water (NFW). Concerning the negative control, one of the reaction mixtures without the DNA template was used. The solution mixture was placed in the Thermal Cycler (Bio-Rad, Hercules, CA, USA) and the PCR cycles parameters were denatured at 94 °C for 5 min followed by 45 cycles of denaturation at 94 °C for 1 min, annealing at 34 °C for 1 min, and polymerization at 72 °C for 2 min. Final elongation was carried out at 72 °C for 7 min. The 1 kb DNA ladder (Vivantis, Selangor, Malaysia) was used as a DNA size marker and fragments were viewed using a UV transilluminator (Syngene, Cambridge, UK).