Skip to main content

Advertisement

Fig. 1 | BMC Biotechnology

Fig. 1

From: A simple, accurate and universal method for quantification of PCR

Fig. 1

Quantification of PCR amplified nucleic acid using AccuCal calibrator. a The workflow associated with using AccuCal to quantify input nucleic acid amount in each PCR, b AccuCal calibrator was diluted so that 0, 40, 60, 80, 120, 140 and 200 ng in Sso Fast EvaGreen Supermix were added to respective wells of a PCR plate and subjected to 40 amplification cycles. c The fluorescence intensity of each AccuCal calibrator (after subtraction of mean 0 ng AccuCal fluorescence) was plotted against the amount (pmols) and a linear regression line fitted to generate the calibration curve. d Ten-fold dilutions of lambda DNA, ranging from 4.5 × 106 – 4.5 × 101 copies/PCR, were amplified in quadruplicate in Sso Fast EvaGreen Supermix on the same plate as the AccuCal calibrators. e The calibration curve was used, alongside the calculated efficiency of each amplification reaction and the cycle numbers between the take-off point (Cq) and second derivative maxima for each amplification reaction, to quantify the mean starting amount of DNA in each PCR. The standard error of the mean is also presented. f The theoretical and determined number of copies/PCR, plus SEM, were plotted against each other and a regression line drawn to demonstrate the agreement between the two values

Back to article page