Dec 05, 2025
qPCR (SYBR Green)
- Vanessa Howland1,
- Yue Ma1
- 1Van Andel Institute
Protocol Citation: Vanessa Howland, Yue Ma 2025. qPCR (SYBR Green). protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zjwrgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 05, 2025
Last Modified: December 05, 2025
Protocol Integer ID: 234300
Keywords: ASAPCRN, qpcr protocol, protocol, sybr
Abstract
qPCR protocol
Materials
- PowerUp™ SYBR™ Green Master Mix (2X)
- Forward and reverse primers (300-800nM)
- cDNA (1-10ng)
- H2O
Troubleshooting
Prepare the reagents
Swirl the PowerUp™ SYBR™ Green Master Mix to mix thoroughly.
Thaw the DNA samples and primers on ice, vortex to mix, then centrifuge briefly.
Set up the PCR reactions
Prepare the appropriate number of reactions, plus 10% overage.
| A | B | C | |
| Component | Volume (10 ul/well) | Volume (20 ul/well) | |
| PowerUp SYBR Green Master Mix (2X) | 5 | 10 | |
| Forward and reverse primers (300-800nM) | |||
| cDNA (1-10ng) | |||
| H20 | |||
| Total | 10 | 20 |
Mix the components thoroughly, then centrifuge briefly to spin down the contents and eliminate any air bubbles.
Transfer the appropriate volume of each reaction to each well of an optical plate.
Seal the plate with an optical adhesive cover, then centrifuge briefly to spin down the contents and eliminate any air bubbles.
PCR can be performed on the reaction plate up to 72 hours after completing the setup, when stored at room temperature. Protect the reaction plate from light if the PCR is not started immediately after the reactions are set up.
Set up and run the real-time PCR instrument
Place the reaction plate in the real-time PCR instrument.
Set the thermal cycling conditions using the default PCR thermal cycling conditions specified in the following tables according to the instrument cycling parameters and melting temperatures of the specific primers. (Easiest is to use the computer software for plate setup)
Standard cycling mode (primer Tm ≥60°C)
| Step | Temperature | Time | Cycles | |
| UDG activation | 50°C | 2 minutes | 1 | |
| Activation (Dual-Lock‱ DNA polymerase) | 95°C | 2 minutes | 1 | |
| Denature | 95°C | 15 seconds | 40 | |
| Anneal | 55–60°C* | 15 seconds | ||
| Extend | 72°C | 1 minute |
Standard cycling mode (primer Tm <60°C), *Anneal temperature should be set to the melting
point for your primers
Set the instrument to perform a default dissociation step.
A dissociation step can be performed up to 72 hours after the real-time PCR run if the plate is stored in the dark and up to 24 hours after the real-time PCR run if the plate is exposed to light.
| Step | Ramp rate | Temperature | Time | |
| 1 | 1.6°C/second | 95°C | 15 seconds | |
| 2 | 1.6°C/second | 60°C | 1 minute | |
| 3[1] | 0.15°C/second | 95°C | 15 seconds |
Dissociation curve conditions (melt curve stage)
Use the following settings for Applied Biosystems™ instruments:
Experiment type: Standard curve or Relative Quantification
Reagent: SYBR™ Green reagents
Reporter: SYBR™
Quencher: None
Passive reference dye: ROX™
Ramp speed: Standard or fast (choose the same setting as in step 2)
Melt curve ramp increment: Continuous
Set the reaction volume appropriate for the type of plate being used for your PCR reaction.
Pay attention to whether you are using a 0.1 or 0.2 ml 96 well plate, the experiment files are not compatible between the two plate sizes.
Start the run.
When the run is finished, make sure to export the results to your USB.