C126EG595: 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 a Bio-Rad iQ5 thermo cycler. The cycle-dependent increase of the fluorescence was quantified at the annealing step (59.5 degrees Celsius).

Usage

data(C126EG595)

Format

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

Details

HPRT1 was amplified in the Bio-Rad iQ5. The the change of fluorescence was simultaneously monitored for the Hydrolysis probe of HPRT1 and EvaGreen. The primer sequences for HPRT1 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 (HPRT1 amplification product), 60 nM hydrolysis probe probe for HPRT1. EvaGreen was used at 0.5 x final. During the amplification was monitored 59.5 degrees Celsius.

Source

Stefan Roediger, Claudia Deutschmann (BTU Cottbus - Senftenberg)

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(C126EG595)
tmp  <- C126EG595

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 \n58.5 degrees Celsius (annealing 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.