This workflow is designed for routine amplification setup where reproducibility, contamination control, and concentration planning matter more than ad hoc bench adjustments.
Steps
- Define reaction volume, primer strategy, and control structure before thawing reagents.
- Calculate master mix composition with enough overage for pipetting loss.
- Confirm template concentration or copy number if the assay needs a standard curve.
- Prepare dilution series in advance rather than adjusting concentration on the fly.
- Aliquot master mix first and add template last.
- Include NTC, positive control, and any standard or reference samples in the same plan.
- Save the final reaction table with lot numbers or reagent versions when relevant.
Control points
- Check whether primer concentrations and total reaction volume match the kit chemistry.
- Keep contamination-sensitive steps physically separated where possible.
- Recalculate any setup if sample concentration changed after re-quantification.
Use these tools
- PCR/qPCR Master Mix
- DNA/RNA Copy Number
- Serial Dilution Planner
Tips
- Document the exact standard range used for qPCR runs.
- Use a calculator before touching reagents when several dilution steps are required.
- Keep a simple one-line summary for successful runs so they can be repeated without guesswork.