Dna thermocycler

RAPD reactions were performed following the process of Williams et al. [16] with some modifications [16] . The screening of primer was done with 15 arbitrary decamer primers (Bengalore Genei, India). Four primers producing good scorable and reproducible bands were selected for subsequent RAPD analysis of bacterial isolates (Table 2). PCR reactions were done in a 10.0-μL reaction mixture containing 1 X PCR buffer (10 mM of tris HCl, pH 8.5; 50 mM of KCl and 15 mM of MgCl 2 ), 10 mM each of dNTP (Bengalore Genei, India), 5 pmol of primer, 2 U of Taq DNA polymerase (Bengalore Genei), 100 ng of genomic DNA. DNA amplification was carried out in a DNA thermocycler (Biometra, Germany) at the following thermal profile: initial denaturation for 3 min at 94 °C, followed by 41 denaturation cycles of 1 min at 94 °C, 1min annealing at 37 °C and extension at 72 °C for 2 min. A final extension step at 72 °C for 10 min was allowed for complete extension of all amplified fragments. Amplified fragments were separated on a 1.5% agarose (Invitrogen, Canada) gel in 1 X TBE buffer along with 20 bp DNA weight marker (Bengalore Genei, India) for 90 min at 100 V. The gel was stained with ethidium bromide (Fisher Scientific) solution (0.1 μg•mL -1 ) for 15 min in a shaker. Finally, the gel was visualized under UVtransilluminator and photographed by Gel Documentation System (Biometra, Germany).

At total of 10 arbitrary 10-mer primers were designed. 29, 30 These primer sequences were as follows: P1: 5′-CCGGCT ACGG -3′, P2: 5′-CAGGCCCTTC-3′, P3: 5′-TACGGAC ACG-3′, P4: 5′-AGCTTCAGGG-3′, P5: 5′-AGGCATTC CC-3′, P6: 5′-GGTCTGAACC-3′, P7: 5′-TAGGCTCA CG-3′, P8: 5′-ACGGTACACT-3′, P9: 5′-GTCCTCAA CG-3′, and P10: 5′-CTTCACCCGA-3′. In a total volume of 25 µL, 50 ng of genomic DNA extracted from carcinoma tissue or corresponding peripheral blood was amplified in 10-mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 50 mM KCl, 200 µM each of dNTP, 0.4 µM of each arbitrary primer, and 1.0 unit of AmpliTaq Gold DNA polymerase (Applied Biosystems, CA, USA). In total, 40 cycles of denaturation (94°C, 30 s), annealing (36°C -40°C, 1 min), and extension (72°C, 2 min) were carried out in a DNA thermocycler (Biometra, Germany). A volume of 5 µL of the PCR products mixed with loading buffer were loaded on to 2% agarose gels and electrophoresed with 100 V for 1 h. The gels were stained with ethidium bromide, and a photograph was taken under ultraviolet light. Loss or gain of band(s) or an increase or decrease in the intensity of the signal of a band (≥50% by image analysis using the Quantity One v 4.4.0 software (Bio-Rad)) in the tumor DNA compared with corresponding peripheral blood DNA was scored as genomic instability.