Journal: BMC Biotechnology

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

doi: 10.1186/s12896-016-0256-y

Figure Lengend Snippet: 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 × 10 6 – 4.5 × 10 1 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

Article Snippet: Serial dilutions of lambda DNA were also amplified as above in either Sso Fast EvaGreen Supermix or in Go Taq hot start colorless mastermix (Promega), the latter being detected with an hydrolysis probe specific to the amplicon and having the sequence 5′ 56-FAM/aacactcaggcacgcggtctg/3IABkFQ 3′ (Integrated DNA Technologies).

Techniques: Amplification, Fluorescence, Lambda DNA Preparation

Journal: BMC Biotechnology

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

doi: 10.1186/s12896-016-0256-y

Figure Lengend Snippet: Quantification using both AccuCal-D and AccuCal-P. a Five, ten-fold dilutions of a known quantity of lambda DNA, ranging from 4.5 × 10 5 to 4.5 × 10 1 , 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 ) 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

Article Snippet: Serial dilutions of lambda DNA were also amplified as above in either Sso Fast EvaGreen Supermix or in Go Taq hot start colorless mastermix (Promega), the latter being detected with an hydrolysis probe specific to the amplicon and having the sequence 5′ 56-FAM/aacactcaggcacgcggtctg/3IABkFQ 3′ (Integrated DNA Technologies).

Techniques: Lambda DNA Preparation, Amplification