Why is there no clear phase separation after demulsification, or why does the aqueous phase appear cloudy? How should this be handled?

Answer:
Under ideal conditions, the aqueous phase should appear clear after demulsification.
Abnormal outcomes are typically caused by incomplete demulsification or contamination (e.g., debris, bubbles).

Possible causes of cloudy aqueous phase:

• Incomplete demulsification (most common):
  Insufficient mixing of the demulsification agent may leave residual droplets suspended in the aqueous phase. 
• Cell debris or contaminants:
  Cell fragments, dead cells, or protein aggregates (e.g., from high BSA concentrations) may remain suspended and cause turbidity. 
• Temperature effects:
  Adding demulsification agent to cold emulsions (e.g., directly from 4°C or after thawing) may impair phase separation. 

Causes of poor phase separation (no clear layering):

• Insufficient demulsification reagent or incubation time 
• Presence of an intermediate emulsion layer (oil–water–debris mixture) 
• Air bubbles interfering with contact between reagent and oil phase 

Recommended actions:

• If incomplete demulsification is suspected:
  Perform a second demulsification step: 
  • Remove the bottom oil phase (avoid removing the aqueous phase) 
  • Add 100 µL Demulsion Agent again 
  • Incubate at room temperature for 2 min 
  • Remove oil phase again 

• If caused by high BSA concentration (>0.04%):
  It may be acceptable to proceed without intervention. 
• If caused by air bubbles during loading:
  Downstream cDNA yield may be reduced; repeating the experiment is recommended. 

Preventive measures:

• Use the correct volume of demulsification reagent 
• Ensure emulsions are equilibrated to room temperature (~25°C) before demulsification 

Avoid introducing bubbles during chip loading