EGFR was amplified from HeLa cDNA using a QuantiTect Primer Assay and the QuantiNova SYBR Green PCR Kit. The template DNA was diluted from 10 ng to 0.01 ng using QuantiNova Yellow Template Dilution Buffer or Tris buffer, and reactions were performed on the [A] Bio-Rad CFX96 and the [B] Applied Biosystems ViiA 7 instruments. Resulting CT values were comparable for both buffers. All reactions were performed in triplicate.
EGFR, ERBB2 and MYC were amplified from HeLa cDNA on the Bio-Rad CFX96, the Applied Biosystems ViiA 7, and the Applied Biosystems StepOnePlus cyclers. Template DNA was diluted in Tris buffer or in QuantiNova Yellow Template Dilution Buffer. Regardless of cycler, cycling conditions, or dilution buffer used, the QuantiNova SYBR Green PCR Kit generates comparable high-quality results.
[A] The single-copy gene IL1R2 was detected in 30, 3, 0.3, and 0.03 ng (10,000–10 target copies) of leukocyte genomic DNA using the QuantiNova SYBR Green PCR Kit to generate a calibration curve for CT value versus number of target copies. The correlation was highly linear. The calibration curve was then used to determine the actual number of target copies in 60 reactions set up to theoretically contain a single copy. [B] Theoretical number of copies expected in each reaction was calculated using Poisson's equation and compared to the actual number of copies determined with the calibration curve. Calculated and experimental frequencies of detection were highly concordant, demonstrating high sensitivity and robustness.
Ten-fold dilutions of HeLa cDNA ranging from 100 ng to 10 fg were quantified on the Applied Biosystems ViiA 7 instrument and the Bio-Rad CFX96 using an in-house assay for beta-actin. [A] The QuantiNova SYBR Green PCR Kit delivers accurate results over a wide dynamic range spanning 8 orders of magnitude. [B] This range is confirmed by the comparable amplification plots from both instruments.
EGFR in HeLa cells was analyzed on various cyclers, fully exploiting the fast-cycling capabilities of each. The QuantiNova SYBR Green PCR Kit provides consistent sensitivity, reproducibility, and efficiency on all tested cyclers, despite varying formats, cycling conditions, and requirement for a passive dye.
QuantiNova DNA Polymerase is kept in an inactive state by QuantiNova Antibody and QuantiNova Guard until the initial heat activation step.
The performance of the QuantiNova SYBR Green PCR Kit was compared to [A] a SYBR Green PCR Kit from supplier B on a Bio-Rad CFX96 cycler and [B] a SYBR Green PCR Kit from supplier L on the Applied Biosystems ViiA 7 cycler. The QuantiNova SYBR Green PCR Kit provides significantly lower CT values, higher reproducibility, and higher reaction efficiency.
The QuantiNova SYBR Green PCR Master Mix contains an inert blue dye. Combined with QuantiNova Yellow Template Dilution Buffer, the resulting solution turns green, indicating that the reaction was set up correctly.