Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 45)

Section 4 Workshop Module
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Programming a Specific Temperature for a Specific Row
To program a specific temperature in any single row:
1. Click Gradient in the Show Options box. Two columns will appear in the spreadsheet and
a representation of the gradient will appear on the right side of the window.
2. Click the Gradient checkbox in the spreadsheet for the desired step.
3. Enter the desired temperature into a specific single row on the gradient display.
4. Press Enter.
5. The temperatures for the other rows will be calculated based on the input desired
temperature and the range.
NOTE: You cannot specify the exact temperature on more than one row at a time.
4.3.5 Melt Curve/Peak
Melt Curve/Peak analysis is a dynamic tool used to measure the melting temperature (T
m
) of
double-stranded DNA (dsDNA) molecules. DNA duplexes can be visualized by either incorporation
of DNA-binding dyes (for example, SYBR
® Green I) or by hybridization with fluorescently labeled
probes. In the case of DNA-binding dyes and non-cleavable hybridization probes, fluorescence is
brightest when the two strands of DNA are annealed. As the temperature is raised towards the
T
m
of the duplex, the fluorescence will decrease at a constant rate (constant slope). At the T
m
,
there is a dramatic reduction in the fluorescence with a noticeable change in slope. The rate of
this change is determined by plotting the negative first derivative (–dF/dT) versus temperature.
The greatest rate changes yield visible peaks, representing the T
m
of the dsDNA complexes.
A melt curve cycle may follow an amplification cycle or can be conducted independently of the
amplification. The melt curve may be programmed in the following manner:
1. Melt curves are programmed as a repeated cycle containing only one step. The
temperature is programmed to increase or decrease incrementally with each repeat of
the cycle. The increase or decrease combined with the number of repeats may not result
in a temperature that is below 4°C or above 100°C at any time during the protocol.
2. Insert a cycle into the protocol at the point that you want the melt curve. The step
generated in this cycle will be used to generate the melt curve data.
3. Enter the temperature at which you wish to begin the melt curve in the Setpoint cell (4–
100°C).
4. Enter an appropriate dwell time for data collection under the Dwell Time column. Dwell
times for the melt curve will vary based on the number of fluorophores in the
experiment. To enter 10 seconds, type 0 followed by a colon (:), then type 10 (that is, as
the time appears in the spreadsheet). Alternatively, 10 seconds can be entered as 0.10.
5. Click in the PCR/Melt Data Acquisition column and select Melt Curve from the pull-down
menu. A green camera will appear in the Data Acquisition cell, and two additional
columns entitled Temperature Change and End Temperature will be added to the
spreadsheet. By default, the temperature change is set at 0.5°C and the end
temperature is at 95°C (unless the setpoint is 95°C). The number of repeats to achieve
this melt curve is automatically calculated. The temperature change can be as low as
0.1°C increments. Typical temperature change values are 0.3–0.5°C for SYBR
® Green I.