Fluorescent Dyes & Oligonucleotide Labeling

NHS-Ester (Amine Coupling)

Best for: 5′/3′ or internal –NH2 modified oligos (e.g., Amino‑C6). Stable amide formation in mildly basic, anhydrous conditions.

• Typical pH 8.3–8.5 (carbonate/borate); avoid primary amines in buffers.
• Examples: FAM‑NHS, TAMRA‑NHS, CF/ATTO/Alexa‑NHS; purify by RP‑HPLC.

Maleimide–Thiol

Best for: 5′/3′‑thiol oligos after deprotection. Rapid thioether formation near neutral pH.

• Reduce disulfides (DTT/TCEP), then desalt before coupling.
• Examples: Cy3‑maleimide, Cy5‑maleimide, ATTO/Alexa‑maleimide.

Click Chemistry

• CuAAC: Alkyne‑oligo + dye‑azide → triazole; fastest but requires copper control for sensitive assays.
• SPAAC: Copper‑free cycloaddition; dye‑DBCO + oligo‑azide; compatible with live‑cell workflows.
• TCO–Tetrazine: Ultra‑fast, copper‑free IEDDA; useful for modular assemblies and smFRET donor/acceptor placement.

Aldehyde‑Reactive (Aminooxy/Hydrazide)

Best for: Oligos bearing aldehyde/ketone (e.g., periodate‑oxidized ribose or formyl linkers). Forms oxime/hydrazone; optional reduction for stability.

Pre‑made Phosphoramidites & Internal Analogues

Best for: Direct on‑column insertion of dye phosphoramidites or internal dye‑dT/dC analogs for controlled spacing/brightness and precise positional labeling.

I. Negative Controls (run in separate tubes)

1. Exogenous Positive Control (run separately)
   What it is: Add external RNA/DNA containing the target (not from your samples).
   Why it matters: Confirms that chemistry, detection, and cycling work and are not inhibited. While a purified amplicon can be used, a synthetic positive control (SPC) with known copy number is more robust for quantitation.
2. Endogenous Positive Control (single-plex or multiplex)
   What it is: A second, native reference gene present in the sample (e.g., housekeeping gene) to normalize and compare fluorescence signals.
   Why it matters: Tracks extraction quality and between-well variability and supports relative quantification.

II. Positive Controls

1. No-Reverse-Transcriptase (No-RT) Control
   What it is: Omit reverse transcriptase during cDNA synthesis.
   Why it matters: Reveals genomic DNA contamination in the RNA prep.
2. No-Template Control (NTC)
   What it is: Omit cDNA (or RNA/DNA) template from PCR.
   Why it matters: Detects reagent/handling contamination and primer-dimer formation. Especially critical with intercalating dyes.
3. No-Amplification Control (NAC)
   What it is: Omit DNA polymerase from PCR.
   Why it matters: Measures background fluorescence of the reaction mix.

Best practice: Run controls in duplicate or triplicate; document expected Cq windows; investigate any control that amplifies unexpectedly (late NTC, positive No-RT, etc.) before interpreting sample results.