Fast SYBR™ Green Master Mix

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.	CR plates, tubes and pipette tips should be UV sterilized for 20-30 mins

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
CR plates, tubes and pipette tips should be UV sterilized for 20-30 mins

Publication protocol

2.5. Real-Time PCR
Real-time PCR for detection of viable bacteria was performed in duplicate using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems) and final volume of 20 μL, containing 0.2 mM of forward and reverse primers, 10 μL of 2 × Fast SYBR Green Master Mix (Applied Biosystems, CA, USA) and 2 μL of prepared DNA template. Primer sequences were (F) AGG GGT TGT ATG CTC GTT GT and (R) TGG AAC ACC TTC AAC TTG CTC T [36], specifically targeting a 121-bp segment of the wzx gene encoding for O antigen flippase of EHEC O157:H7. Cycling conditions included an initial activation step at 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 3 s, and annealing temperatures at 60°C for 30 s. After 40 PCR cycles, melting curves were generated at steps of 95°C for 15 s, 60°C for 1 min, followed by 95°C for 30 s, and 60°C for 15 s. Threshold cycle (C t) values were automatically generated by the 7500 Fast Real-Time PCR software. In all cases, negative and positive controls of amplification were included using 2 μL nuclease free water (Applied Biosystems, CA, USA) or known genomic DNA isolated from R508N, respectively.

To quantify the number of E. coli O157:H7 cells in samples, a standard curve was prepared from each plate in triplicate for each DNA concentration. Standard curves were generated by amplifying a DNA dilution series of a known number of E. coli O157:H7 cells.



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Manufacturer protocol

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