High CV from Bubbles in Wells

Symptom

Inconsistent absorbance readings between replicate wells with coefficient of variation >15%. Visual inspection may reveal bubbles present in wells, particularly when using detergent-containing solutions.

1. 1 Solutions containing detergents are especially susceptible to bubble formation
2. 2 Rapid or forceful addition of reagents to wells introduces air
3. 3 Bubbles remaining in wells interfere with optical reading path
4. 4 Multi-channel pipettors may trap air when dispensing detergent solutions

1. 1 Ensure no bubbles are present prior to reading the plate by visual inspection
2. 2 Gently add solutions to the wells to minimize turbulence and air incorporation
3. 3 Pop bubbles by touching them with a dry pipette tip before reading
4. 4 Allow plate to stand 1-2 minutes after reagent addition for bubbles to dissipate

Related Video (2)

Bilibili (China-Accessible Mirrors) ★ 72

ELISA Operation Demonstration Video

"Concise hands-on ELISA benchwork video where proper pipetting/well-handling technique matters most for avoiding the bubble artifact."

Thermo Fisher Scientific ★ 70

How to Run an ELISA Assay – Invitrogen Kit Step-by-Step Tutorial

"Step-by-step kit tutorial with plates and reagents visible, useful for seeing where detergent solutions and bubbles arise in the workflow."