Ptc 200

Because we inserted human fibroblasts and bFGF into the nude mice, the gene expression of collagen type I in the implanted materials was examined using RT-PCR with human-specific primers. The results were normalized to the mRNA level of human GAPDH.
Total RNA was extracted by adding 0.5 ml of TRIzol® reagent (Invitrogen, Life Technologies, USA) to N₂-frozen nude mice tissues. Each μg of RNA was subjected to cDNA synthesis by using SuperScript™ Reverse Transcriptase II (Invitrogen) and oligo (dT)12–18 primers (Invitrogen) in a 20 μl reaction volume according to the manufacturer’s instructions, with the additional step of removing the RNA complementary to the cDNA using E. coli RNase H (Invitrogen). One microliter of each cDNA was then subjected to polymerase chain reaction (PCR) according to the following amplification profile: predenaturation at 94 °C for 40 s, amplification (denaturation at 94 °C for 40 s; annealing at 60 °C for 40 s; extension at 72 °C for 1 min) for 30 cycles, and a final extension at 72 °C for 10 min in a DNA thermal cycler (model PTC-200, MJ Research, Inc., MA, USA). For each of the PCR products, 10 μl was electrophoresed on a 1.5% agarose gel in the presence of ethidium bromide and visualized by the Gel Documentation System (Vilber Lourmat, France).

Genes ctxB and tcpA of randomly selected V. cholerae O1 strains in 2012 were sequenced following conditions as described elsewhere [34 (link)]. PCR amplification of genes ctxB and tcpA was performed in a 25 μl reaction mixture in an automated Peltier thermal cycler (PTC-200, M. J. Research). Subsequently, PCR products were purified with a Microcon centrifugal filter device (Millipore Corporation, Bedford, MA) and sequenced using an ABI PRISM Big Dye Terminator Cycle Sequencing Reaction kit (Applied Biosystems, Foster City, CA) on an ABI PRISM 310 automated sequencer (Applied Biosystems). The deduced amino acid sequences of the respective genes from all strains were aligned using CLUSTAL-W.

For PCR analyses two different primer pairs were used (1-1697for and 1-1697rev; 271-1290for and 271-1290rev) (Table 1). The PCR reaction mix was prepared in a total volume of 50 μl, and the Expand High Fidelity PCR System (Roche, Mannheim, Germany) was employed. 100 ng genomic DNA or cDNA and 50 pmol per primer were used. Thermal cycling was carried out in a thermocycler PTC-200 (MJ Research, Bio-Rad Laboratories, Hercules, USA) using the following settings: 3 min 94°C; 30 times: 30 sec 94°C, 30 sec 57°C, 70 sec 72°C; 7 min 72°C; ∞ 4°C. PCR products were loaded on 1% agarose gel (Lonza, Rockland, USA) and the gel was documented by Gel Doc XR⁺ (Biorad, Hercules, USA).