R: Helicase Dependent Amplification of HPRT1 with different...
C67
R Documentation
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
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.
http://www.ncbi.nlm.nih.gov/pubmed/22437246