This protocol is for the typical ligation of DNA done in the Richard Lab.The consensus Ligation protocol should be consulted if deviating from the procedure outlined here.
1.Mix together the following:
- a. 5µl digested front part (EcoRI and SpeI).
- b. 5µl digested rear part (XbaI and PstI).
- c. 4µl deionized water.
- d. 3µl digested vector (EcoRI and PstI).
- e. 2µl of Reaction Buffer for T4 Ligase.
- f. 1µl of DNA Ligase.
2. Incubate at room-temp for 10 mins (or at 4°C overnight)
3. Kill Reaction for 15 mins at 80°C
- Mike has had problems with blunt end ligation if this reaction is let go for too long.
- If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.