What should I do if no clear phase separation is observed after demulsification? (No transparent oil phase / no pink aqueous phase)

Possible Causes:

• Insufficient Demulsion Agent (less than 100 μL per sample), leading to incomplete breaking of the water-in-oil emulsion.
• The emulsion product was not equilibrated to room temperature before adding the Demulsion Agent. Temperature differences between the cold (4 °C) product and room temperature reagent may affect phase separation.
• Presence of excessive air bubbles during droplet generation, which prevents the Demulsion Agent from properly interacting with the oil phase.

Recommended Solutions:

1. Add more Demulsion Agent and allow to incubate again:
   1. Ensure 100 μL Demulsion Agent is added per sample.
   2. Let the tube stand at room temperature for 2 min without shaking.
   3. Observe until clear separation is visible: the upper transparent oil layer (Demulsion Agent/Carrier Oil) and lower pink aqueous phase (reaction mixture).
   4. If clear separation still does not form, the emulsion structure may be abnormal—discard the sample and repeat from Step 1 (Emulsion Generation and Reverse Transcription).

2. Ensure temperature equilibration before demulsification:

• After reverse transcription, remove PCR tubes from the thermal cycler and leave them at room temperature for 1 min before adding the Demulsion Agent. This prevents temperature shock that could interfere with phase separation.

3. Avoid air bubbles during droplet generation:

• After each loading step, confirm that no bubbles remain before proceeding to the next step. Preventing bubbles at the source will improve demulsification efficiency.