Amplification Failure Due to Inadequate Reaction Mixing

Symptom

Reactions show inconsistent color change or partial amplification with some replicates failing, caused by reagent stratification or incomplete mixing before incubation.

1. 1 Master mix components not properly mixed after thawing, causing uneven distribution of polymerase, primers, or dNTPs
2. 2 Reaction tubes not mixed after adding template, leaving nucleic acid concentrated at bottom or sides
3. 3 High-viscosity samples (e.g., crude lysates) create density gradients preventing proper mixing
4. 4 Cold reagents added directly to room-temperature master mix causing temperature stratification

1. 1 Vortex all master mix components for 5-10 seconds after thawing, then briefly centrifuge to collect contents
2. 2 Mix each assembled reaction by pipetting up and down 5-8 times or pulse vortex 3-5 seconds before incubation
3. 3 Bring all reagents to room temperature before combining to minimize density differences
4. 4 Briefly centrifuge reaction tubes (2-3 seconds) after mixing to eliminate air bubbles and ensure liquid is at bottom
5. 5 For viscous samples, dilute 1:2 to 1:5 with nuclease-free water before adding to reactions