You may be experiencing pipetting errors
Pipetting errors cause inconsistent delivery of reagents or sample to the wells, leading to:
- Trailing clusters
- Some (or all) samples clustering with the NTCs
- Cloudy or diffuse clusters
- A sample (or samples) does not cluster with a specific allele (i.e. is an outlier)
Improve pipetting precision by:
- Calibrating and cleaning pipettors regularly
- Pipetting larger volumes (no less than 5 µL) for greater accuracy and precision
- Reducing the number of pipetting steps whenever possible
- Increasing the consistency of the pipetting method (such as using robotic pipetting)
- Consulting the manufacturer about the correct method of dispensing liquid volumes accurately from the pipettor. For example, some pipettors are designed to deliver the designated volume at the first plunger stop, so "blowing out" the residual volume may cause error.
Validate the pipetting process by preparing a replicate plate (same assay and sample across the plate) to ensure results are reproducible.