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The iCycler iQ and Amplifluor Detection Systems for Analysis of Genetically Modified Organisms in Foodstuffs, Rev A

by 50 cycles of 95C for 15 sec, 55C for 20 sec, and 72C for 40 sec. Fluorescent data were collected during the 55C step.

Determination of GMO Content
Primers were chosen to amplify part of the construct used in Roundup Ready GM soy. The following master mix was prepared:

90 l 10x PCR buffer (Life Technologies)
90 l dNTPs (2.5 mM)
90 l forward primer (4 M)
90 l reverse primer (directly labeled with UniPrimer sequence and energy transfer system) (4 M)
9 l Platinum Taq polymerase (5 U/l, Life Technologies)
27 l MgCl2 (50 mM)
432 l H2O (tissue-culture grade, Life Technologies)

DNA and controls and the 96-well plate were prepared as above and run using the same thermal cycling protocol.


Results
Determination of Relative Soy DNA Content
Background-corrected data were brought down to the PCR baseline. The standard curve generated by the in-house standards showed a correlation coefficient (r) of 0.991 (Figure 1). The correlation coefficient was not improved by extension of the number of cycles to be included in the baseline. The data showed that soy DNA could be quantitated at all dilutions used (Figure 2), and it was possible to calculate the amount of soy DNA in the commercial standards relative to the in-house standards.

Determination of GMO Content
Background-corrected data were brought down to the PCR baseline. The standard curve generated by the in-house standards showed a correlation coefficient (r) of 0.996. This could be improved to 0.998 when the number of background cycles was increased to 22 (Figure 3). The data showed that GM s
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