Team:UCL London/Ligation
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Latest revision as of 20:47, 24 October 2010
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.