Resolution:
## Figure 2.
Assessing the impact of SYBR Green I quantity on real-time amplification profiles. Replicate amplification reactions were prepared using three commercial enzyme formulations
supplemented with increasing amounts of SYBR Green I. Each amplification reaction
contained 100 femtograms of lambda gDNA (1,876 genomes) and 500 μM of the primers
K7B and K12. The gain setting for each run was adjusted to ensure that reaction fluorescence
remained below the saturation level of the photomultiplier tube (about 40,000 FU).
(A), (C) and (E) Screen shots of amplification profiles generated by the Stratagene MxPro qPCR software.
This reveals that fluorescence intensity is dependent on SYBR Green I quantity, reflected
by the large increase in the height of the amplification profiles as SYBR Green I
quantity is increased. (B), (D) and (F) LRE plot of each respective amplification profile. This reveals a linear domain corresponding
to the central region of each amplification profile, confirming the mathematical prediction
that amplification efficiency is linearly coupled to amplicon quantity. A consecutive
group of points was selected (designated by red circles) for linear regression analysis
(referred to as "LRE analysis"), generating estimates for E_{max }(intercept) and ΔE (slope), from which F_{max }was calculated using equation 4 (see Figure 1 for additional details). Details as
to how the boundaries of the linear region were determined are described later in
the study. [SG]: quantity of supplementary SYBR Green I, r2: linear regression correlation coefficient.
Rutledge and Stewart |