Real-time PCR was run on TECHNE QUANTICA (Duxford, Cambridge, UK), using the following programme, UNG incubation at 50°C for 5 min, initial denaturation at 95°C for 15 min followed by cycling (45 cycles) denaturation at 95°C for 10 s, annealing at 64°C for 20 s, extension at 72°C for 30 s, and final extension for 7 min at 72°C.
Thermal cycling was performed in a Thermocycler using the following programme 5 min at 95°C, 30 × (30 sec at 95°C, 30 sec at 65°C, 60 sec at 72°C), 5 min at 72°C.
Amplification was performed using the following programme: 95°C for 1 minute; 35 cycles of 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds; 72°C for two minutes.
Thermal cycling was performed in a Thermocycler using the following programme: 10 min at 95°C, 35× (30 sec at 96°C, 30 sec at 60°C, 60 sec at 72°C).
The real-time PCR reaction was carried out in a Light Cycler 480 II (Roche Life Science) using the following programme: Preincubation 95 °C for 10 min, 45 cycles of 95 °C 10 s, 60 °C 30 s, 72 °C 1 s and a cooling step at 40 °C fro 30 s.
The samples were run on a GeneAmp 5700 Sequence Detector Real-Time PCR machine (Applied Biosystems) using the following programme: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min with products being detected using SYBR Green or the fluorescent Taqman probe.
A PCR amplification system (PTC-100: MJ, USA) was used with the following programme: initial denaturation for 4 min at 94°C, 30 s at 94°C, 30 s at 56°C, 30 s at 72°C, 20 cycles; 7 min at 72°C.
Chromo4 real-time PCR machine (Biorad) was used with the following programme: 95 °C for 10 minutes, 95 °C for 10 seconds, 60 °C for 15 seconds, 72 °C for 15 seconds, 75 °C for 1 second, plate read, repeat from the second step 39 more times, 72 °C for 5 minutes, melting curve from 55 °C to 95 °C, read every 0.2 °C, hold for 1 second and finally 75 °C for 5 minutes.
The initial multiplex PCR was performed in a 25 µl reaction volume containing 1× PCR buffer, 6.5 mM MgCl2, 600 µM of each dNTP, 0.01 µM of each primer, and 2 U of AmpliTaq Gold® DNA polymerase (AB) using the following cycling programme: denaturation at 94°C for 5 minutes followed by 35 cycles at 95°C for 30 sec., 60°C for 30 sec., and 65°C for 30 sec., followed by 6 minutes at 65°C.
PCR was carried out using the following thermal cycler programme: 94°C 5 min; 30 cycles of 94°C 30 s, 43.5°C 45 s, 72°C 2 min; final extension 72°C 7 min. Fingerprints were performed from biological duplicates.
PCR was performed using the following thermal cycler programme: 94°C 5 min; 30 cycles of 94°C 30 s, 52°C 45 s, 72°C 2 min; final extension 72°C 7 min. PCR products were purified using E.Z.N.A. Cycle-Pure kit (Omega Bio-Tek, Doraville, GA, USA).
