This workflow links DNA quantitation, molar-ratio planning, and dilution setup so ligation decisions are documented before reagents are mixed.
Steps
- Confirm vector and insert sizes from the intended construct map.
- Quantify DNA and review whether purity is adequate for ligation.
- Use copy-number or concentration tools when stock values are unclear.
- Calculate insert and vector amounts with the ligation setup tool.
- Prepare any intermediate dilutions needed to avoid sub-microliter pipetting.
- Include vector-only and no-ligase controls where background matters.
- Record the exact molar ratio, buffer lot, and incubation condition.
Control points
- Recheck DNA lengths before calculating molar ratio.
- Avoid using mass ratio as a substitute for molar ratio.
- Confirm that salts or carryover from cleanup will not inhibit ligase.
Use these tools
- Ligation Setup
- Multi-Stock Mixing
- DNA/RNA Copy Number
Tips
- Prepare a small planning table before moving to the bench.
- If any component volume is too small, make an intermediate dilution first.
- Save the final setup used for the successful construct to improve reproducibility.