One of the perks about the Open qPCR interface is how simple and quick it is to create complex experiments. In this post, we’ll run through the basic elements for setting up a real-time PCR protocol.
This is the initial screen you’ll see once logged in through the browser. You’ll want to select Create a New Experiment to proceed to the thermal protocol settings.
After you’ve entered your experiment name, you’ll be brought to the protocol settings page to define the exact parameters for your assay. The default settings are shown in the image below.
To set up your protocol, you’ll need to specify the following parameters:
• Total number of steps and stages
• Temperature, ramp speed, and hold duration for each step
• Data acquisition step(s)
EDIT STEPS + STAGES
We’ll start off the protocol setup with adding steps and stages.
First, let’s define a step. A step contains a specific temperature and time frame at which the reaction will be held. There is no limit to the number of steps that may be added into your experiment protocol.
A stage, on the other hand, may contain one or more steps. You may add as many steps as needed under a stage. Once you select the Add Stage icon, you’ll have three options.
The default protocol comes with a holding stage that contains the initial denaturation step. Additional steps may be added to a holding stage if needed. Generally, in both two-step and three-step PCR protocols, a holding stage exists at the very beginning since it houses the initial denaturation step.
In some cases, depending on whether you plan to run downstream applications such as a post-PCR agarose gel, an additional holding stage may be added at the very end for the final extension step.
The cycling stage is like a holding stage that repeats. It is essentially where all the good stuff happens - your target DNA sequence amplifies here. Denaturation, annealing and extension occurs during this stage so the number of DNA copies doubles with each completed cycle. If you happen to specify 35 cycles, you’ll have ~34 billion copies of template DNA in your end reaction!
Melt Curve Stage
Melt curve analysis is performed to confirm primer specificity as well as any potential primer-dimers in the reaction. It is oftentimes also used to identify PCR results for genotyping purposes. The melting temperature (Tm) of a PCR product is defined as the temperature that corresponds to the peak maximum of that product. The melt curve Tm is the temperature at which 50% of DNA has denatured from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA). Melt curve plots are displayed as the negative first derivative (-dF/dT) versus temperature.
A melt curve is typically performed immediately after the amplification protocol in one continuous sitting. The below illustration shows a sample amplification protocol followed by a melt curve.
Add a Pause
When a pause is implemented, the reaction will be held for an indefinite amount of time at a set temperature. This option is useful if you need to perform a two-step reverse transcriptase PCR (RT-PCR) protocol.
The Edit Stages button is where you can delete and reposition any step in the protocol.
To delete a step, select the x icon. To reposition a step in the protocol, click and drag the dotted icon at the bottom of the orange section.
Under this section, you can specify the temperature, ramp speed and hold duration for each step.
With each new experiment you create, the ramp speed between every step is set to Auto by default. Auto is defined as the instrument’s maximum ramp speed for both heating and cooling. The ramp speed refers to the speed at which the heat block changes temperature between adjacent steps.
Depending on your assay requirements, you can adjust ramp speeds to anywhere between 0.00001 and 5° C/s. If applicable, you can designate different ramp speeds between each step. Since ramp speed affects the time length it takes to complete a specified number of cycles, a 5° C/s ramp speed can significantly decrease the overall assay time.
For some users who want to perform downstream activities post-amplification, an infinite hold may be added at a specified temperature. This option is only available during the last step with the Gather Data feature turned off.
To implement this feature, set the Hold Duration to 00:00.
The nice thing about the Open qPCR software is that you can gather data during any step or ramp.
When you create a new experiment, the default protocol is a 2-step PCR set to gather data at the end of the run (indicated by the amplification curve icon in the image below). While data is generally acquired during the extension step of each cycling stage, this ultimately depends on the PCR chemistry you design your assay around. For instance, in the case of both SYBR Green and Hydrolysis Probes (aka TaqMan Probes), fluorescent signal is optimal at the end of the extension step. However, for Molecular Beacon-based detection, fluorescent signal should be gathered during the annealing step or before the extension step begins for each PCR cycle.
If you plan to run only an endpoint reaction with no fluorescence detection (aka conventional PCR), make sure to turn off Gather Data for Step and/or Ramp in all stages of your protocol. You will not receive amplification curves with end-point reaction settings.
Once you have your protocol set, click on Start Experiment and your targets are on their way to amplification!
As a side note, you probably noticed that we did not address the AutoDelta feature (aka Touchdown PCR) here. There are some specific details to cover so we’ll get to this in a later post.
If you have any questions or comments, let us know by posting below!