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One microliter of the multiplexed PCR products was added to 9 μl of either 2% 400 HD [ROX] sizing standard (Applied Biosystems) or 2% 500 [ROX] sizing standard (Applied Biosystems) depending on the estimated sizes of products being analyzed.
Additionally, peak overpressure predictions are compared against empirical correlations and the NFPA 68 vent sizing standard.
The two batches were fragment analyzed on an ABI3130 genetic analyzer with the Genescan LIZ600 (Applied Biosystems Inc., Foster City, CA, USA) sizing standard.
The PCR products were electrophoresed on a LI-COR 4200 automated sequencer along with a fluorescently labelled size standard (50-350 bp sizing standard; LI-COR® Biotechnology Division).
Four lanes of sizing standard [50 350 bp in length and labeled with appropriate infrared dye (purchased from Li-Cor)] were included on each run and each gel was scored using the computer software saga (version 3.3; Li-Cor, Lincoln, NE, USA).
Genomic DNA, in agarose inserts [ 56], was electroeluted into a solution containing a lambda DNA sizing standard (30 pg/μl), and then were mounted on cleaned, derivitized glass surfaces using a microfluidic device [ 57] followed by polymerization of a thin layer of polyacrylamide (3.3% containing 0.02% Triton X-100).
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