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Molecular biology

Restriction digest and ligation cloning

Cut vector and insert with restriction enzymes, dephosphorylate the vector, purify and ligate at a set insert-to-vector molar ratio.

Total time3–4 h (or overnight ligation)
Hands-on1 h
LevelIntermediate
Steps7
0 of 7 steps done

Before you start

  • Check enzyme buffer compatibility for double digests and methylation sensitivity.

Reagents and materials

Totals are for 2 digests (+10% extra). Per-digest (50 µL) amounts are in the middle column.

ReagentPer digest (50 µL)Total
DNA (vector or insert)1 µg1 µg
10× restriction buffer5 µL5 µL
Restriction enzyme(s), 10–20 U each1 µL1 µL
Nuclease-free waterto 50 µL43 µL43 µL

Buffers and recipes

Ligation (20 µL) per reaction

Dephosphorylated vector50 ng
Insert (3:1 molar ratio)see formula
10× T4 ligase buffer (contains ATP)2 µL
T4 DNA ligase1 µL
Nuclease-free waterto 20 µL

Insert (ng) = vector (ng) × insert length (bp) ÷ vector length (bp) × molar ratio.

Equipment

  • 37 °C heat block
  • Gel extraction or PCR clean-up kit
  • Thermocycler or 16 °C block

Procedure

Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.

  1. Digest

    Assemble 50 µL digests and incubate at 37 °C for 1 h (time-saver enzymes: 5–15 min).

  2. Dephosphorylate the vector

    Add phosphatase (e.g. rSAP or CIP) to the vector digest for 30 min at 37 °C, then heat-inactivate as instructed.

  3. Purify

    Run on an agarose gel and extract the correct bands (or column-clean the insert PCR digest).

  4. Quantify

    Measure DNA concentrations and calculate insert amount for a 3:1 ratio.

  5. Ligate

    Assemble ligation and incubate 10 min–1 h at room temperature or overnight at 16 °C. Include a vector-only control.

  6. Transform

    Transform 2–5 µL into competent cells; heat-inactivate the ligase first if you electroporate.

  7. Screen

    Pick colonies for colony PCR or miniprep and diagnostic digest; confirm by sequencing.

Critical points

  • Avoid UV damage when extracting from gels (use blue light).
  • Total glycerol in a digest should stay below 5% to avoid star activity.

Troubleshooting

ProblemWhat to check or change
Many empty-vector coloniesDephosphorylate longer, gel-purify the vector, check single-cut background.
No coloniesCheck ATP in ligase buffer (avoid freeze-thaw), insert ratio, competent cells.

Safety

Standard laboratory care; UV protection for gel extraction.

Further reading

  • New England Biolabs technical reference: restriction digest and ligation guidelines.

This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.