This workflow is for the common situation where you have freshly measured nucleic acid concentration and need to decide whether the sample is clean enough, whether it needs dilution, and how it should be carried into a final PCR setup.
Steps
- Measure concentration and purity using the same blank and instrument conditions you will record in the notebook.
- Decide whether the sample is acceptable for downstream PCR or whether cleanup is needed first.
- If the sample is usable but too concentrated, design the dilution before touching PCR reagents.
- Confirm the final working concentration required for the assay.
- Build the PCR master mix with the correct control wells and enough overage.
- Add template after the shared mix is balanced and aliquoted.
Control points
- Do not let a good concentration number override a suspicious purity profile.
- Recalculate any dilution if the sample is re-measured after cleanup.
- Confirm that the final template input matches the assay design, not just the stock concentration.
Use these tools
- A260 Quantitation
- Serial Dilution Planner
- PCR/qPCR Master Mix
Tips
- Record the exact purity decision so later failures can be traced back to sample quality.
- If the dilution plan requires tiny transfers, create an intermediate stock first.
- Treat cleanup, dilution, and PCR setup as one documented chain rather than three disconnected steps.