Your pipetting may be inaccurate
Pipetting errors can cause inconsistent delivery of reagents or sample to the wells, resulting in:
- Trailing clusters
- Some (or all) samples clustering with the NTCs
- Cloudy or diffuse clusters
- A sample (or samples) not clustering with a specific allele (i.e., it is an outlier)
Improve pipetting precision by following these guidelines:
- Calibrate and clean the pipettors regularly.
- Pipette larger volumes (no less than 5 µL) for greater accuracy and precision.
- Reduce the number of pipetting steps whenever possible.
- Increase the consistency of the pipetting method (i.e., robotic pipettors).
- Consult 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 technique by preparing a cocktail with the PCR reagents, assay and sample. Pipette equal volumes into all wells of the plate and determine if the results are reproducible.