C126EG685: qPCR Experiment for the Amplification of HPRT1 Using the...

In chipPCR: Toolkit of Helper Functions to Pre-Process Amplification Data

Description

A quantitative PCR (qPCR) with the DNA binding dye (EvaGreen) (Mao et al. 2007) was performed in the Roche Light Cycler 1.5 thermo cycler. The cycle-dependent increase of the fluorescence was quantified at the elongation step (68.5 degrees Celsius).

Usage

data(C126EG685)

Format

A data frame with 40 observations on the following 97 variables. The first column ("Cycles") contains the number of cycles and consecutive columns contain the replicates ("A01" to "H12").

Details

MLC-2v was amplified in the Roche Light Cycler 1.5. The the change of fluorescence was simultaneously monitored for the Hydrolysis probe of MLC-2v and EvaGreen. The primer sequences for MLC-2v were taken from Roediger et al. (2013). A 10 micro L qPCR reaction was composed of 250 nM primer (forward and reverse), qPCR Mix (according to the manufactures recommendations), 1 micro L template (MLC-2v amplification product), 60 nM hydrolysis probe probe for MLC-2v. EvaGreen was used at 0.5 x final. The amplification was monitored at 68.5 degrees Celsius (elongation step).

Source

Claudia Deutschmann & Stefan Roediger, BTU Cottbus - Senftenberg, Senftenberg, Germany

References

A Highly Versatile Microscope Imaging Technology Platform for the Multiplex Real-Time Detection of Biomolecules and Autoimmune Antibodies. S. Roediger, P. Schierack, A. Boehm, J. Nitschke, I. Berger, U. Froemmel, C. Schmidt, M. Ruhland, I. Schimke, D. Roggenbuck, W. Lehmann and C. Schroeder. Advances in Biochemical Bioengineering/Biotechnology. 133:33–74, 2013.

Mao, F., Leung, W.-Y., Xin, X., 2007. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol. 7, 76.

Examples

data(C126EG685)
tmp <- C126EG685

plot(NA,NA, xlim = c(1,40), ylim = c(min(tmp[, 2:ncol(tmp)]), 
    max(tmp[, 2:ncol(tmp)])), xlab = "Cycle", 
    ylab = "RFU (FAM)", 
    main = "Amplification monitored at \n68.5 degrees Celsius (elongation 
step)")

apply(tmp[, 2:ncol(tmp)], 2, 
      function(x) lines(tmp[1:nrow(tmp),1],x))
      

chipPCR documentation built on March 5, 2021, 9:06 a.m.