Scientific 1d imaging system

Total RNA was isolated from cell specimens using the guanidinium thiocyanate-phenol method. The extract integrity was assessed by 1.5% agarose gel electrophoresis, and RNA was visualized by ethidium bromide staining. The total amount of RNA was determined spectrophotometrically. RT-PCR assay was performed according to De Petro et al. [17 (link)] with slight modifications. An aliquot of total RNA (1 μg) was use for RT. The RT product (2 μL) was diluted with the PCR buffer (50 mM KCl, 10 mM Tris-HCl, and 2 mM MgCl₂) to a final volume of 50 μL, containing 0.5 μM dNTPs (final concentration, 0.8 mM) and 0.5 unit of Super-Therm Taq DNA polymerase (Southern Cross Biotechnology, Cape Town, South Africa). PCR was performed on a GeneAmp PCR system 2400 (Applied Biosystems, Foster City, CA). The ODN primers used in RT-PCR were as described previously [18 (link)]. The PCR products were analyzed by 1.5% agarose gel electrophoresis and direct visualization after SYBR Green I (Cambrex Bio Science Rockland, Inc., Rockland, ME) staining. The agarose gel was scanned and analyzed using the Kodak Scientific 1D Imaging System (Eastman Kodak Company, New Haven, CT).

PCR of urease A and cag A genes of H pylori from gastric juice was used to measure the reality of H pylori infection status. PCR was well performed with the purified genomic DNA as the template. The sequence information of primers including urease A and cag A virulence factors was listed in the Table 1. DNA extracted product from gastric juice (40 ng) was diluted with the PCR buffer (50 mM KCl, 10 mM Tris–HCl, and 2 mM MgCl2) to a final volume of 20 mL, containing 0.5U of Taq DNA polymerase (Invitrogen), 0.25 μM dNTPs master mix (Yeastern Biotech, Taiwan) and 0.5 μM primer. For each experiment, up to 60 cycles were performed in a Bio-Rad thermal cycler (Bio-Rad). PCR was initiated with a hot start (1 minute at 95°C), the samples were then subjected to 60 cycles at 95°C for 1 minute, 48°C for 1 minute, and 72°C for 1 minute. The PCR products (urease A: 414 bp, and cag A: 349 bp) were analyzed on 2% agarose gel electrophoresis and the Kodak Scientific 1D Imaging System (Eastman Kodak Company, New Haven, CT).