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UCL London/Protocol/Ligation
From 2009.igem.org
- Materials:
- Plasmid DNA: 15 µL
- Restriction Enzymes (depend on the requirements of the parts)
- EcoRI: 1 µL
- PstI: 1 µL
- SpeI: 1 µL
- XbaI: 1 µL
- 10× NE buffer: 1.5 µL/1 µL (lid)
- BSA: 1 µL (lid)
- Quick Ligase
- 2× Quick Ligation Buffer
- Method:
- Digest the DNA as in Restriction Enzyme Digestion Protocol.
- Heat inactive the restriction enzymes.
- Run a diagnostic gel before the ligation.
- After the gel is confirmed, add 1 µL quick ligase, 10 µL ligation buffer, 3 µL backbone and 6 µL inserts (or other volumes depend on the concentration of DNA) into an eppendorf.
- Leave for 5 minutes (or up to 30 minutes) at room temperature (25°C).
- Transform the ligated DNA onto competent cells as described in Transformation, method 2 step 4 to 10.
