( Methods Determination of R...)The iCycler iQ and Amplifluor Detection Systems for Analysis of Genetically Modified Organisms in Foodstuffs, Rev A

Methods
Determination of Relative Soy DNA Content
A master mix was prepared to quantitate the relative amounts of soy DNA in commercially available and in-house Roundup Ready GM soy standards. Primers were chosen to amplify part of the soy lectin gene. The master mix contained:

90 l 10x PCR buffer (Life Technologies)
90 l dNTPs (2.5 mM)
90 l lectin gene forward primer (2.5 M)
90 l lectin gene reverse primer (including recognition sequence) (0.5 M)
90 l Amplifluor UniPrimer primer (5.0 M, Intergen)
9 l Platinum Taq polymerase (5 U/l, Life Technologies)
27 l MgCl₂ (50 mM)
342 l H₂O (tissue-culture grade, Life Technologies)

The master mix was thoroughly mixed and stored on ice prior to use. DNA was prepared from an in-house Roundup Ready GM soy standard and diluted to 20 ng/l to make a 100% soy stock. This stock was then further diluted in a 10-fold dilution series to a 0.1% soy stock. A 2 l aliquot of each diluted soy DNA stock was placed in each of 3 wells on a 96-well optical plate. DNA was also prepared from commercially available Roundup Ready GM soy standards to use as unknowns. Standards were obtained from Fluka (series 410R) and containined 0%, 0.1%, 0.5%, 1%, 2%, and 5% Roundup Ready GM soy. DNA from each of the commercial standards was diluted to 20 ng/l and 2 l of these stocks was added to each of 3 wells on the same 96-well plate. Three control reactions containing 2 l of tissue-culture grade water were also prepared. To each tube containing DNA or water, 23 l of master mix was then added. The plate was covered with a sheet of optical sealing film. PCR conditions were 95C for 3 min, followed
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