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. 9µl of digested gel el extracted insert
- b. 5µl of digested gel extracted vector
- c. 2µl of Reaction Buffer (or any NEBuffer)
- d. 2µl of 10mM ATP solution (only if NEBuffer was used)
- e.1µl of DNA Ligase
- f. 1µl of PEG solution (optional)
- g. Water to bring up to 20µl
2. Incubate at room-temp for 30-120 mins (or at 16°C overnight) 3. Kill Reaction for 15 mins at 70°C
- Mike has achieved good results with leaving the ligation in a drawer overnight, but this should not be done if using PEG to speed up the ligation.
- If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.