This workflow links the cloning sequence from ligation planning through transformation review. The goal is not only to assemble a reaction, but also to preserve enough information to tell whether failure came from ligation, competent cells, or plating conditions.
Steps
- Confirm vector and insert lengths and recent DNA concentrations.
- Calculate the planned ligation ratio and check whether transfer volumes are realistic.
- Prepare any intermediate dilutions needed to avoid sub-microliter transfers.
- Include the right controls, especially vector-only or positive controls where relevant.
- Transform the ligation and the controls under comparable conditions.
- Record recovery volume, plated fraction, and any dilution prior to plating.
- Calculate transformation efficiency if the outcome needs to be interpreted quantitatively.
Control points
- Distinguish ligation problems from competent-cell problems using controls.
- Keep the actual plated fraction in the notes or later efficiency calculations become unreliable.
- Recheck fragment lengths after any construct revision.
Use these tools
- Ligation Setup
- Multi-Stock Mixing
- Bacterial Transformation Efficiency
Tips
- If one stock is too concentrated, fix the pipetting problem before the ligation tube is built.
- Write the control design before starting, not after the plates come back weak.
- Compare ligation and control outcomes only if the plating logic was consistent.