Restriction Enzyme Based Cloning
- If insert is PCR, run PCR then PCR purify (Qiagen kit). Elute in 30 uL EB
- Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C.
- Gel purify both vector and insert (Qiagen kit).
- Run out on gel (see Preparing an Agarose Gel)
- On a gel box cut out appropriate bands and place in clean eppendorf tubes. Try to limit the exposure to UV light
- Weigh out gel piece. For each mg add 3 uL of QG (orange) buffer. For example, for a 0.1g piece add 300 uL buffer.
- Place at 55-65C until gel is dissolved (5-10 min)
- Add to pink purification column, wash and elute in 30 uL
- Combine 6 uL insert with 1 uL vector. Place at 50-65C for 5-10 min to help sticky end binding.
- Add 2 uL ligase buffer (single use aliquots).
- Add 1 uL T4 DNA Ligase (Invitrogen).
- Incubate 2h-O/N at 16C (water bath in cold room).
- Transform 5-10 uL into 50 uL supercompetent cells:
- Make 50 uL aliquots
- Add DNA and mix gently
- Incubate on ice for 30min
- Heat shock for 45s at 42C
- Place on ice for 2min
- Add 450 uL LB
- Incubate at 37C for 1h with shaking
- Plate 500 uL and grow O/N at 37C on appropriate antibiotic plates
- Pick several colonies and digest to verify insert. Sequence if necessary.