C67 {chipPCR}

Helicase Dependent Amplification of HPRT1 with different input DNA quantities using the Bio-Rad iQ5 thermo cycler

Description

A Helicase Dependent Amplification (HDA) of HPRT1 (Homo sapiens hypoxanthine phosphoribosyltransferase 1) was performed at three different input DNA quantities using the Bio-Rad iQ5 thermo cycler. The HDA was performed at 65 degrees Celsius. The optimal temperature for a HDA is circa 65 degrees Celsius. Lower temperatures will affect the slope and plateau of the HDA amplification curve.

Usage

data(C67)

Format

A data frame with 43 observations on the following 6 variables.

Cycles.C67

a numeric vector containing the cycle numbers

t.C67

a numeric vector containing the time elapsed between the cycles. The time was calculated by the cycle duration of one iQ5 thermocycler step (71 seconds / step).

D1

Dilution 1.

D2

Dilution 2.

D3

Dilution 3.

D4

Dilution 4.

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 started directly after adding buffer B and the preincubation step. Temperature profile if the iQ5 thermo cycler (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(C67)
matplot(C67[, -c(1,2)], type = "l", xlab = "Time [sec]", ylab = "RFU")