Description
Probe master mix for SNP genotyping
GenieQ Genotyping Probe Master Mix is a 2X probe-based master mix optimised for allelic discrimination and SNP genotyping. It gives clear cluster separation between genotypes. Robust endpoint and real-time performance supports reliable dual-probe (e.g. FAM/VIC) assays across common platforms.
Just add primers, TaqMan MGB probes and template. An optimised Taq DNA polymerase and buffer raise the genotyping success rate on low-concentration and complex templates. The mix accepts 1–10 ng template input, genotypes SNP sites with 25–73% GC content accurately, and can amplify directly from blood lysate. A specific ROX passive reference dye works on all qPCR instruments without adjusting its concentration. The mix amplifies genomic DNA, cDNA, plasmid DNA and λDNA templates. For research use only.
Key Features
| Blood lysate compatible | SNP genotyping can be performed with blood lysate alone as the template. | |
| Broad input range | Compatible with template inputs of 1–10 ng. | |
| GC-rich tolerance | Accurately genotypes SNP sites with a GC content of 25–73%. | |
| Universal ROX reference dye | Suits all qPCR instruments with no ROX adjustment. |
Specifications
| Format | 2X probe master mix |
| Application | SNP genotyping / allelic discrimination by probe-based qPCR |
| Detection chemistry | Dual TaqMan MGB probes with endpoint signal acquisition |
| Template input | 1–10 ng gDNA per 20 μl reaction |
| SNP GC content | 25–73% |
| Reference dye | Universal ROX passive reference dye (no adjustment needed) |
| Shelf life | 2 years |
Components
| Components | Amount |
| GenieQ Genotyping Probe Master Mix (2X) | 500 rxns |
Reaction numbers are based on 20 μl reactions.
Storage
Store at -30 ~ -15°C.
- Prepare the PCR reaction
| Components | Volume |
| GenieQ Genotyping Probe Master Mix (2X) | 10 μl |
| Primer F (10 μM) | 1.8 μl |
| Primer R (10 μM) | 1.8 μl |
| TaqMan MGB Probe A (10 μM) | 0.4 μl |
| TaqMan MGB Probe B (10 μM) | 0.4 μl |
| gDNA | 1–10 ng |
| ddH2O | Up to 20 μl |
- For convenience, combine primers and probes into a 20X assay (e.g. 18 μl 100 μM Primer F, 18 μl 100 μM Primer R, 4 μl 100 μM Probe A and 4 μl 100 μM Probe B, made up to 100 μl with TE). Recommended final concentrations are 900 nM for each primer and 200 nM for each probe.
- Do not use ROX-labelled probes; the mix already contains ROX.
- Obtain primers and probes from a commercial TaqMan genotyping assay or design them with dedicated software such as Primer Express.
- Include no-template controls (NTCs) and positive controls of known genotype in every run.
- If the reaction cannot be run immediately, store mixed samples in the dark at 2 ~ 8°C for up to 72 h.
- Run the PCR program
| Stage | Temperature | Time | Cycles |
| Initial denaturation | 95°C | 30 sec | 1 |
| Denaturation | 95°C | 10 sec | 45 |
| Annealing/extension | 60°C | 30 sec | 45 |
| Endpoint signal acquisition | 60°C | 30 sec | 1 |
- Acquire the endpoint signal and analyse results
- If the endpoint signal cannot be read immediately after PCR, store samples in the dark at 2 ~ 8°C for up to 72 h.
Q1: What should I do if there is no signal or a low signal?
- Check the template: confirm DNA integrity by agarose gel, re-measure the DNA concentration, dilute the template to reduce inhibitors, or increase the template input or cycle number. Check the reagents: use a new batch if expired, seal wells to prevent evaporation and read signals promptly, and confirm that primers, probes, template and mix are all in the wells. Check by BLAST that no SNP sites fall within the primer sequences and redesign if needed. Finally, confirm the correct reporter channel is selected and re-read the endpoint signal.
Q2: Why are the signals too scattered to form clusters?
- Inhibitors in the template (dilute it), too little template (increase input or cycle number), the wrong reporter channel (correct it and re-read), or ROX not selected on an instrument that needs ROX correction.
Q3: Why are the genotype clusters too close together?
- The template may be degraded (check by agarose gel); probes may be degraded (repeat with a new batch and check storage of primers, probes and mix); the probe Tm may be outside the optimal range; or too many cycles were run (do not exceed 45).
Q4: Why is clustering poor with tailing signals?
- Re-measure the DNA concentration so inputs are consistent within 1–10 ng and dilute the template if inhibitors are present; use fresh reagents, seal wells and read promptly; make sure all components are in each well and mixed thoroughly; calibrate the instrument regularly; and select ROX on instruments that require ROX correction.
Q5: Why is the no-template control signal too high?
- The reagents may be contaminated: replace primers, probes, master mix and all consumables and repeat. The instrument may also have fluorescent contamination and should be cleaned.
