biotechrabbit™ CAPITAL™ qPCR Probe Master Mix Plus is a high-performance solution designed for quantitative PCR (qPCR) applications that require utmost precision and reliability. This ready-to-use mix combines a robust Taq DNA polymerase, UDG (Uracil-DNA Glycosylase), and dUTP to provide enhanced protection against carryover contamination, ensuring the accuracy and reproducibility of your qPCR results.
The UDG enzyme is incorporated to degrade uracil-containing DNA, a common byproduct of PCR carryover, while the inclusion of dUTP ensures that any contaminating DNA from previous PCRs is not amplified. This system not only improves the reliability of qPCR assays but also simplifies the workflow by preventing false positives.
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Component |
Composition |
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4X CAPITAL™ qPCR Probe Master Mix UDG |
Optimized 4X qPCR Probe Master Mix with UDG for prevention of carryover contamination. |
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Storage |
Store at −20 °C until expiry date. |
Handling
Prevention of PCR contamination
When assembling the amplification reactions, care should be taken to eliminate the possibility of contamination with undesired DNA.
- Use separate clean areas for preparation of samples and reaction mixtures and for cycling.
- Wear fresh gloves. Use sterile tubes and pipette tips with aerosol filters for PCR setup.
- Use only water and reagents that are free of DNA and nucleases.
- With every PCR setup, perform a contamination control reaction that does not include template DNA.
Basic Protocol
- Aliquot the master mix to minimize freeze-thaw cycles and light exposure.
- Thaw on ice and mix all reagents very well. Assemble and keep all reactions on ice.
- Use only high-quality, optically clear reaction plates and seals designed for fluorescence applications.
- Do not use corner wells, or use a more robust seal.
- Reserve plate positions for positive controls (control DNA) and negative controls (water or buffer).
- First pipette the primer-probe mixture, then add the template, and add the Master Mix last.
- Before preparing mixes, calculate the volume needed according to the number of reactions plus one extra reaction.
- For better correlation, run the reactions in triplicate.
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Component |
Volume |
Final concentration |
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Primer Mix (Reverse and Forward) |
Variable |
100–400 nM |
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Too high primer concentrations result in unspecific amplification and should be avoided. |
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|
Specific Probe |
Variable |
200 nM |
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Template DNA |
Variable |
10 pg–100 ng |
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Use diluted or undiluted cDNA from less than 1 µg RNA. |
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CAPITAL™ qPCR Probe Mix, 4× |
5 µl |
1× |
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Nuclease-free water |
Variable |
|
|
Total volume |
20 µl |
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- Gently mix the reactions without creating bubbles. Do not vortex. Bubbles will interfere with fluorescence detection.
- Place the reaction into the PCR cycler.
Cycling program
|
Step |
Temperature |
Time |
Cycles |
|
Initial activation |
95 °C |
2–3 min |
1 |
|
Denaturation |
95 °C |
10–15 s |
40–45 |
|
Annealing/Extension* |
60–68 °C |
30 s |
*Recommended annealing temperature is 2 °C above primer Tm. Use gradient PCR to optimize the annealing temperature. Do not use temperatures below 60 °C.
Do not exceed 30 seconds. For melt analysis, refer to the instrument instructions.
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