Frequently Asked Questions
FAQ
1. Should the kit be stored at -20 °C?
Store polymerase at -20 °C and 2x buffer at 22 °C.
2. How does TrueLAMP suppress nonspecific amplification?
The buffer contains a proprietary inhibitor formulated with the polymerase to prevent nonspecific primer amplification when no template is present.
3. What incubator works best?
Uniform heating is critical to avoid evaporation and condensation in 10 µL reactions. Recommended: Fully submerged water bath or temperature-controlled smart coffee mug (POC) or thermocycler with heated lid. Evenly air-circulated digital oven may also work. Do not use heat bloc.
4. Why is my NTC positive?
1) Recalibrate pipettes. Incorrect volumes (< 4.75 µL buffer per 10 µL reaction) may reduce inhibition. 2) Verify incubation temperature. Nonspecific amplification can occur at ≤ 63 °C. 3) Increase buffer slightly (e.g., 5.25 µL 2× buffer per 10 µL reaction) for stubborn primer sets.
5. Why did my positive control fail?
Template concentration may be below LOD. Determine LOD for each primer set.
6. Can raw samples (e.g., saliva, plasma) be used?
Purified DNA/RNA is recommended. TrueLAMP is sensitive to pH and ionic strength. Water-suspended cells or virions may be compatible after validation.
7. Can polymerase be premixed with 2× buffer for storage?
No. Polymerase activity declines rapidly when premixed. Prepare reaction mix fresh.
8. Why is a 61 °C pre-incubation needed and how long should reactions run?
An initial 61 °C 5-10 min pre-incubation followed by 65 °C for 50-80 min improves amplification consistency and robustness across DNA and RNA targets, LAMP primer sets, and incubation systems (oven, bath, thermocycler). Amplification is generally visible by 30 min and reaches maximum by 90 min. However, strict endpoint timing is not required. Extended incubation does not typically alter endpoint color.
9. How should color be quantified?
Use a smartphone colorimeter app. Sample the region just below the liquid meniscus with the smallest aperture and record: minimum green (RGB mode) or maximum magenta (CMYK mode).