A profile is a group of Programs that will instruct the instrument to heat up and cool down depending on what experiment you are running in the instrument.
Select the Profile tab. Since this is a new experiment, the profile is empty - there are no programs displayed, and the Temperature Profile is blank as shown below.
This display shows the programs you have added to the Programs list in the order they will occur during the experiment. The order in which they appear in the Temperature Profile corresponds to the order they have been added to the Programs list. The Temperature Profile displays how long the experiment will take on the x axis as well as time stamps after every program. These timestamps are displayed as vertical lines with an annotated time stamp.
All profiles are made up of programs, which are run by the instrument in the order they are displayed. Each program performs a role within the experiment, like holding a temperature, cycling to perform a PCR, or performing a temperature ramp to measure melting peaks.
Add a Hold for pre-incubation by selecting Add, selecting Hold from the Select Predefined Program window and click Select.
The hold has now been added to the programs list and is present in the temperature profile window.
A cycle program will now be added by selecting a 2-Step Amplification program from the Choose Predefined Program window. Once selected it has been added to the temperature profile window as shown below.
The cycling program defines a phase of the experiment where the instrument will repeatedly heat and cool to a defined series of temperatures. Each repeat is called a cycle.
The Cycling pane allows you to edit the settings of the program. Name can be edited as for all phases, and is displayed in the Programs list.
No. of Cycles
No. of Cycles sets how many times the cycle should be repeated, for example 30, 45 or 60 times. A table displays the settings for each stage of a cycle. Each row of the table gives the settings for a single stage of the cycle.
The default settings as displayed are for two stages, where the first stage will ramp to 95C at 5C/s and hold for 10s, then the second stage will ramp to 60C at 4C/s and hold for 30s before taking an optical acquisition.
Clicking on a cell in the Acquire column will cause an optical acquisition to be carried out after that stage. Note that only a single stage can be selected for optical acquisition.
Cycling Stage Editing
Note that the stages in a Cycling Phase can be edited by adding and deleting elements, and moving elements up and down in the list. These controls work the same way as for editing phases in the Phases list, as covered in this section. All list controls are standardised and are also described in the section Editing Lists.
A melting program will be added from the choose predefined program window which updates the temperature profile as follows.
A melt phase defines a phase of the experiment where the instrument will ramp to an initial temperature, then ramp to a final temperature. While ramping to the final temperature, optical acquisitions will be made continuously. These can then be analysed to yield melt peaks.
Once again the Name of the phase can be edited in the Melting pane*.*
For both the initial and final stages, you can edit the Temp. at which the hold is performed, the Ramp rate at which the instrument moves to the hold temperature, and the Hold time in seconds, using the corresponding controls in the Melting pane.
First, we will delete the existing Cycling Phase - just select this phase in the Phases list, and click the Delete button at the bottom of the Phases list. The Cycling Phase will disappear.
Now click the Add button, and select a 3-Step Touchdown Phase to be added.
The settings for a 3-Step Touchdown program are exactly the same as for a normal Cycling program, but with the addition of a Touchdown Settings area at the bottom of the pane, and an extra column in the stages table, titled T.D.
A touchdown program allows you to specify that one of the stages of each cycle will have its target temperature modified as the cycling proceeds. This allows for the early cycles of a PCR to have a higher temperature specified, leading to more specific amplification. The stages should be set up as normal, with the standard temperatures specified - for example in the default touchdown phase we have added above, annealing is performed at 65C. Note that the T.D. Column is ticked for this stage - this means that the annealing stage will perform a touchdown, varying the target temperature on each cycle. To select a different stage to perform the touchdown, just click the checkbox in the T.D. column for that stage. Only one stage can perform touchdown in each touchdown program.
In the Touchdown Settings area, we can now specify the increase in temperature on the first cycle of the run - by default this is set to 5C, giving a temperature of 65C + 5C = 70C for the first cycle. On each subsequent cycle, the temperature is moved towards the normal value of 65C, dropping by a specified temperature difference each cycle - by default this is 1C per cycle. When enough cycles have passed that the temperature has reached the normal value, it will remain at the normal value for the remaining cycles.
Fused Project Profiles
When viewing the profile in a fused project the software will give you a profile summary of the profiles that are contained in the separate experiments. In the example below two HRM experiments have been fused that contained different HRM melt programs.
The Profiles pane gives a list of the profiles present in the fused project. Whichever profile has been selected in this list will appear in the Temperature Profile pane. The profile that has not been selected has been greyed out as can be seen in the Temperature Profile above.
Additional information about the different profiles can be seen in the Programs and Details pane on the bottom right of the software window.