Accu prep pcr dna purification kit

Using the ITS1 and ITS4 primers, The ITS1 and ITS2 and the inverting 5.8S coding rDNA were amplified [81 (link)]. In a total volume of 50 µL, each PCR reaction mixture comprised 5–10 ng genomic DNA, 1 µM of each ITS1/ITS4 primers, 5 µL of a 10X reaction buffer (50 mM KCl, 50 mM Tris–HCl; pH 8.3, 0.1 mg/mL bovine serum albumin (BSA), 3 mM MgCl₂, 200 µM each of dNTP, and 2.5 U of Taq DNA polymerase (Promega, Mannheim, Germany). The sequences of the ITS1 and ITS4 primers are; 5 ‘-TCCGTAGGTGAACCTGCGG-3 ‘and 5′ TCCTCCGCTTATTGATAT GC-3′, respectively. The PCR technique includes 35 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, and elongation at 72 °C for 1 min. Before DNA sequencing, the PCR amplicon was resolved using a 8% agarose gel and purified using a specific PCR purification kit (Accu Prep® PCR DNA Purification Kit, K-3034–1, Bioneer Corporation, South Korea). MacrogenInc, (South Korea) sequenced the purified PCR products. All inter transcribed spacer sequencing work was also performed by MacrogenInc, (South Korea) and was carried out on both strands of the submitted DNA fragments. The purified PCR products were sequenced using an ABI 377 DNA. Auto sequencer (PerkinElmer, Applied Biosystems Div., Waltham, USA) based on the same primers mentioned before.

The ITS1 and ITS2 as well as the inverted 5.8S coding rDNA were amplified using the ITS1 and ITS4 primers. In a total volume of 50 µL, each PCR reaction mixture comprised 5–10 ng of genomic DNA, 1 µM of each ITS1/ITS4 primer, 5 µL of a 10X reaction buffer (50 mM KCl, 50 mM Tris–HCl; pH 8.3, 0.1 mg/mL bovine serum albumin (BSA), 3 mM MgCl₂, 200 µM each of dNTP, and 2.5 U of Taq DNA polymerase (Promega, Mannheim, Germany). The PCR technique includes 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and elongation at 72°C for 1 min. Before DNA sequencing, the PCR amplicon was resolved using an 8% agarose gel and purified using a specific PCR purification kit (Accu Prep® PCR DNA Purification Kit, K-3034–1, Bioneer Corporation, South Korea). MacrogenInc, (South Korea) sequenced the purified PCR products. All inter-transcribed spacer sequencing work was also performed by MacrogenInc, (South Korea) and was carried out on both strands of the submitted DNA fragments [121 (link)]. The purified PCR products were sequenced using an ABI 377 DNA. Auto-sequencer (PerkinElmer, Applied Biosystems Div., Waltham, USA) based on the same primers mentioned before.