Luna® Universal qPCR Master Mix Protocol (M3003)
- Prepare DNA or cDNA of interest using desired DNA extraction and purification method.
- Make dilutions of DNA or cDNA to be used for the standard curve. These should be prepared fresh before each experiment and can be diluted in either water or TE.
Reaction Setup: For best results, we recommend running each DNA standard and sample in triplicate.
| COMPONENT | 20 µl REACTION |
FINAL CONCENTRATION |
| Luna Universal qPCR Master Mix |
10 µl |
1X |
| Forward primer (10 µM) |
0.5 µl |
0.25 µM |
| Reverse primer (10 µM) |
0.5 µl |
0.25 µM |
| Template DNA |
variable | < 100 ng |
| Nuclease-free Water |
to 20 µl |
- Thaw Luna Universal qPCR Master Mix and other reaction components at room temperature, then place on ice. After thawing completely, briefly mix each component by inversion, pipetting or gentle vortexing.
- Determine the total volume for the appropriate number of reactions, plus 10% overage and prepare assay mix of all components except DNA template accordingly. Mix thoroughly but gently by pipetting or vortexing. Collect liquid to the bottom of the tube by brief centrifugation.
- Aliquot assay mix into qPCR tubes or plate. For best results, ensure accurate and consistent pipetting volumes and minimize bubbles.
- Add DNA templates to qPCR tubes or plate. Seal tubes with flat, optically transparent caps; seal plates with optically transparent film. Care should be taken to properly seal plate edges and corners to prevent artifacts caused by evaporation.
- Spin tubes or plates briefly to remove bubbles and collect liquid (1 minute at 2,500–3,000 rpm).
- Program real-time instrument with indicated thermocycling protocol (see table below). Ensure a plate read is included at the end of the extension step.
Use the SYBR® or SYBR/FAM scan mode setting on the real-time instrument.
We recommend using the “Fast” ramp speed where applicable (e.g., Applied Biosystems StepOnePlus®, QuantStudio®, 7500 Fast instruments).
| CYCLE STEP |
TEMPERATURE |
TIME | CYCLES |
| Initial Denaturation |
95°C | 60 seconds |
1 |
| Denaturation Extension |
95°C 60°C |
15 seconds 30 seconds (+plate read) |
40-45 |
| Melt Curve |
60-95°C* | various | 1 |