Geldoc uv transilluminator

A multiplex PCR was developed with the aim to identify the six species of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri and L. welshimeri) and Listeria spp., by using the PCR protocol described by Ryu et al. (2013 ) that has been partially modified in order to obtain the seven differentials bands for each Listeria species in a single reaction. Primers concentrations are reported in Table 1. All amplification reactions were performed in a final volume of 25 µL containing 5 µL of DNA, 5 µL of 10X PCR buffer (JumpStart REDTq DNA Polymerase, Sigma-Aldrich, St. Louis, MO, USA), 4 mM of MgCl₂, 0.1 mM each of dNTP, and 2 U of JumpStart RED Taq (Sigma-Aldrich). All amplification reactions were performed in a Gene-Amp 2700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) programmed as follows: denaturation at 94°C for 5 min, annealing at 58°C for 30 sec and elongation at 72°C for 30 sec, followed by a final extension period at 72°C for 5 min. The amplified fragments were separated by 3% agarose gel electrophoresis (Roche diagnostics, Milan, Italy) in 1X Tris-acetate EDTA (TAE; Invitrogen, Carlsbad, CA, USA) and stained with ethidium bromide (0.1 mg/mL) for 20 min. The gels were observed and the images acquired by the Gel-Doc UV trans-illuminator (Bio-Rad, Hercules, CA, USA).