CD75: Helicase Dependent Amplification in the 'VideoScan' 'HCU'

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

Description

Helicase Dependent Amplification in the 'VideoScan' 'HCU' of HPRT1 (Homo sapiens hypoxanthine phosphoribosyltransferase 1)

Usage

data(CD75)

Format

A data frame with 93 observations on the following 6 variables. The data frame contains three replicates of a HDA for HPRT1.

CD75.t1

Elapsed time during HDA in seconds.

CD75.F1

Time-dependent fluorescence during HDA.

CD75.t2

Elapsed time during HDA in seconds.a numeric vector

CD75.F2

Time-dependent fluorescence during HDA

CD75.t3

Elapsed time during HDA in seconds.

CD75.F3

Time-dependent fluorescence during HDA.

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 12.5 micro L buffer A containing 1.25 micro L 10x reaction buffer, 150 nM primer (forward and reverse), 0.75 micro L template (synthetic) and A. bidest which was covered with 50 micro L mineral oil. The primer sequences for HPRT1 were taken from Roediger et al. (2013). Preincubation: This mixture was incubated for 2 min at 95 degree. Celsius and immediately placed on ice. 12.5 micro L of reaction buffer B which was composed of 1.25 micro L 10x buffer, 40 mM NaCl, 5 mM MgSO4, 1.75 micro L dNTPs, 0.2 x EvaGreen, 1 micro L Enzyme mix and A. bidest. The fluorescence measurement in 'VideoScan' 'HCU' started directly after adding buffer B at 55, 60 or 65 degrees Celsius and revealed optimal conditions for the amplification when using 60 or 65 degree Celsius. 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(CD75)
matplot(CD75[1:80, c(1,3,4)], CD75[1:80, c(2,4,6)], type = "l", 
        xlab = "Temperature", ylab = "RFU")

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