C81: Helicase Dependent Amplification of pCNG1 using the...

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

Description

A Helicase Dependent Amplification (HDA) of pCNG1 was performed. The 'VideoScan' Platform (Roediger et al. (2013)) was used to monitor the amplification. The HDA was performed at 65 degrees Celsius. Two concentrations of input DNA were used.

Usage

data(C81)

Format

A data frame with 351 observations on the following 5 variables.

Cycle

Cycles HDA measurements.

t.D1

Dilution 1, elapsed time during HDA in seconds.

MFI.D1

Dilution 1, fluorescence.

t.D2

Dilution 2, elapsed time during HDA in seconds.

MFI.D2

Dilution 2, fluorescence.

Details

To perform an isothermal amplification in 'VideoScan', standard conditions for the IsoAmp(R) III Universal tHDA Kit (Biohelix) were used. The reaction was composed of reaction mix A)10 micro L A. bidest, 1.25 micro L 10xbuffer, 0.75 micro L primer(150 nM final), 0.5 micro L template plasmid. Preincubation: This mixture was incubated for 2 min at 95 degree. Celsius and immediately placed on ice. Reaction mix B) 5 micro L A. bidest., 1.25 micro L 10x buffer, 2 micro L NaCl, 1.25 micro L MgSO4, 1.75 micro L dNTPs, 0.25 micro L EvaGreen, 1 micro L enzyme mix. The mix was covered with 50 micro L mineral oil. The fluorescence measurement in 'VideoScan' 'HCU' started directly after adding buffer B at 65 degrees Celsius. A 1x (D1) and a 1:10 dilution (D2) were tested. Temperature profile (after Preincubation): - 60 seconds at 65 degrees Celsius - 11 seconds at 55 degrees Celsius && Measurement

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.

Examples

data(C81)
# First example
# Comparison of Lowess, Moving average and splines to smooth amplification curve 
# data of
# HDA for pCNG1.

plot(NA, NA, xlim = c(0, 120), ylim = c(0, 1.2), xlab = "Time [min]", 
     ylab = "Fluorescence", main = "VideScan HCU HDA amplification - Raw data")
  points(C81[, 2]/60, C81[, 3], type = "b", col = 1, pch = 20)
  points(C81[, 4]/60, C81[, 5], type = "b", col = 2, pch = 20)
legend(2000, 0.4, c("D1", "D2"), col = c(1,2), pch = rep(20,2))

Example output

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