5-VIC Phosphoramidite

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.2 Concentration recommendation: 10 μM.

2. Working Solution Preparation
2.1 Diluent: Nuclease-free water (alternatively DNase/RNase-free water).
2.2 Working concentration: 0.05 μM, 500 nM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type: Nucleic acid samples (reverse-transcribed cDNA, recombinant plasmid templates, genomic DNA from bacterial samples, DNA extracted from spiked samples)^[1][2]:
3.2 Incubation conditions:
3.2.1 For qPCR-based assays[1]: Use 0.05 μM 5-VIC Phosphoramidite labeled probe; perform amplification cycles with denaturation at 95°C for 10 s, annealing at 50.6°C for 10 s, and extension at 72°C for 20 s, for a total of 40 cycles, preceded by an initial preincubation at 95°C for 10 s.
3.2.2 For droplet digital PCR (ddPCR) assays[2]: Include 500 nM 5-VIC Phosphoramidite probe in the ddPCR reaction mixture; perform thermal cycling with the program: 60°C for 5 min, followed by 40 cycles of 95°C for 20 s and 60°C for 30 s.
3.3 Washing steps: No washing steps specified for PCR-based assays^[1][2].

4. Controls
4.1 Include blank control (water as template), negative control, and positive control.
4.2 Blank control excludes reagent fluorescence interference.
4.3 Positive control validates experimental system.

5. Detection & Analysis
5.1 Instrument type: Quantitative real-time PCR instrument, droplet digital PCR system with fluorescence detection capability.
5.2 Ex/Em wavelengths: VIC channel fluorescence signals.
5.3 Result analysis:
5.3.1 For qPCR-based assays[1]: Fluorescence intensity increases with successful amplification of target nucleic acid templates; positive samples exhibit a sigmoidal amplification curve, with cycle threshold (Ct) values correlating with template concentration (lower Ct values indicate higher template load); positive results are indicated by VIC channel fluorescence.
5.3.2 For ddPCR assays[2]: Positive samples show distinct green fluorescence amplitude separation from negative droplets; positive fluorescence signals indicate the presence of the target sequence; positive reactions exhibit green fluorescence.