UCL London/Protocol/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.