Positive Control Remains Pink After Incubation

Symptom

The positive control reaction fails to turn yellow following 30-minute incubation at 65°C, indicating no amplification occurred despite presence of target template.

1. 1 Improper pipetting technique during reaction setup resulted in insufficient master mix or primer addition
2. 2 Master mix components were not thoroughly mixed after thawing, causing uneven distribution of enzymes or dNTPs
3. 3 Reactions were not adequately mixed prior to incubation, leaving stratified layers
4. 4 Incubation temperature did not reach or maintain 65°C throughout the reaction period
5. 5 Enzyme activity compromised by freeze-thaw cycles or improper storage temperature

1. 1 Use calibrated pipettes and verify proper technique with volume checks before setting up reactions
2. 2 Thoroughly vortex and briefly centrifuge all reagents after thawing to ensure homogenous mixing
3. 3 Mix each reaction by gentle pipetting 5-8 times or brief vortex before placing in incubator
4. 4 Verify incubator temperature with independent thermometer; preheat heating block to 65°C before adding tubes
5. 5 Minimize freeze-thaw cycles by aliquoting master mix into single-use volumes; store at -20°C