Ligation

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

1. Digest the DNA as in Restriction Enzyme Digestion Protocol.

2. Heat inactive the restriction enzymes.

3. Run a diagnostic gel before the ligation.

4. 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.

5. Leave for 5 minutes (or up to 30 minutes) at room temperature (25°C).

6. Transform the ligated DNA onto competent cells as described in Transformation, method 2 step 4 to 10.