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Fig. 2 | BMC Biotechnology

Fig. 2

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

Fig. 2

Quantification using both AccuCal-D and AccuCal-P. a Five, ten-fold dilutions of a known quantity of lambda DNA, ranging from 4.5 × 105 to 4.5 × 101, were amplified twice in quadruplicate and detected using either EvaGreen, the intercalating dye in Sso Fast mastermix, or a FAM-labelled hydrolysis probe specific to the target amplicon. In both cases, AccuCal-D and AccuCal-P calibrators were included on the same plate and used to independently quantify the starting amount of input DNA in each PCR. The theoretical amount versus the calculated amount, determined by either AccuCal-D or AccuCal-P, using the EvaGreen dye (EG) or the hydrolysis probe (P), was plotted and the linear regression of each is shown in the graph on the right. b Five, ten-fold dilutions of a known quantity of lambda DNA in quadruplicate were amplified in Sso Fast mastermix using primers to give a 501 bp amplicon. AccuCal-D and AccuCal-P calibrators were included on the same plate and were used to independently quantify the starting amount of lambda DNA in each PCR. The theoretical amount versus the calculated amount, determined by either AccuCal-D or AccuCal-P, for the 501 bp amplicon, was plotted and the linear regression of each is shown in the graph on the right. c Five, ten-fold dilutions (3 one-hundred fold dilutions on the Eco) of a known quantity of lambda DNA were amplified in various mastermixes (see Methods) on the different qPCR platforms indicated over 2–10 PCR runs. The theoretical amount versus the mean calculated amount, determined by AccuCal-D, across all platforms was plotted and the linear regression is shown in the graph on the right. The mean number of calculated copies/PCR and SEM are shown in each case

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