A second set of primers named NEST-F 5′-GGAAACCCTGATGCAGCA-3′ and NEST-R 5′-ACACAGTTTTATATGCTT-3′ annealing www.selleckchem.com/products/DAPT-GSI-IX.html within the amplicon obtained by the primers CAF and CAR were designed to attest and improve first PCR protocol sensitivity. Primers were tested using the amplify 3 program (Bill Engels, University of Wisconsin, 2005). PCR assays were carried out in a reaction mixture containing 10 mmol L−1 Tris-HCl, 50 mmol L−1 KCl, 200 μmol L−1 of each dNTP, 0.2 μmol L−1 of each primer, 1.5 mmol L−1

MgCl2, 1.25 UI TaqGold Polymerase (Applera Italia, Monza, Italy) and 2 μL of DNA (100 ng μL−1) in a final volume of 50 μL. Thermal cycler conditions consisted of 95 °C denaturation for 5 min, 30 cycles of 95 °C for 1 min, 57 °C for 1 min, 72 °C for 1 min and a final extension at 72 °C for 7 min in a Thermal Cycler (DNA Engine Dyad peltier Thermal Cycler, BioRad, Milan, Italy). To

verify the specificity of the CAF and CAR primers for the C. arthromitus DNA sequence, the PCR protocol was tested on the microorganisms listed in Table 1. On the basis of the data reported by Spanggaard et al. (2000), mostly Gram-negative bacteria were chosen for the test. The bacteria were grown overnight at 30 °C on brain–heart infusion agar (Oxoid), and a single colony was picked from the plate and transferred into a 1.5-mL microfuge tube containing 0.3-g glass beads and DNA extraction was performed as

described by Manzano et al. (2003). Primers CAF and CAR Selleckchem Ibrutinib were used for PCR on the DNA extracted from fish gut. It was not possible to know the Idelalisib research buy detection limit of the DNA concentration needed in order to obtain a positive result in the PCR assay because of the unculturable nature of the microorganism This led us to utilize heterogeneous DNA from fish as a template. For this reason, a nested PCR to attest the specificity of the positive result achieved and to decrease the detection limit of the protocol was prepared. As PCR product visualization is difficult when the DNA concentration loaded in the agarose gel is below 20 ng, the new primers NEST-F 5′-GGAAACCCTGATGCAGCA-3′ and NEST-R 5′-ACACAGTTTTATATGCTT-3′ were used in a second PCR step (nested PCR). The PCR assay was performed in a reaction mixture containing 10 mmol L−1 Tris-HCl, 50 mmol L−1 KCl, 200 μmol L−1 of each dNTP, 0.2 μmol L−1 of each primer, 1.5 mmol L−1 MgCl2, 2.5 UI TaqGold Polymerase (Applera Italia) and 2 μL of PCR product in a final volume of 50 μL. The thermal cycler conditions consisted of 95 °C denaturation for 5 min, 30 cycles of 95 °C for 1 min, 45 °C for 1 min, 72 °C for 1 min and a final extension at 72 °C for 7 min in a Thermal Cycler.

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