DNA cloning requires the DNA sequence of interest to be inserted in a vector
DNA molecule. For this, both the vector as well as insert DNA is prepared by
digestion with compatible restriction enzymes, so that the ends produced during
digestion is complementary in both. When setting up ligation, it is important
to consider the permutations that can occur and bias the relative concentration
of DNA accordingly.
Usually a 5- to 10-fold excess of insert over the vector DNA is the norm.
This ensures that enough ligated product will be produced in the right orientation.
- Vector digest
- Insert DNA
- T4 DNA ligase
- Ligation buffer: 50 mM Tris-HCI, 10 mM MgCI2, 20 mM DTT, 1 mM ATP,
- Nuclease-free BSA, pH 7.4 (generally supplied with the enzyme)
- Set up ligation in 0.5 mL microfuge tubes as follows:
- Voetoriligest (DHa.5).
- Insert DNA.
- Ligation buffer.
- Mix well by flicking the tube.
- Spin briefly in a microfuge to push all the liquid to the bottom of the tube.
Incubate at 16°C overnight.
- Stop the reaction by placing it at 70°C for 10 minutes.
- Run an aliquot on a minigel and verify.
The ligation buffer supplied by the vector, along with the enzyme, usually
contains ATP. However, one should verify this and in case there is not ATP or
if the buffer is old, it should be supplemented with ATP.
The incubation temperature in step 4 depends on the ends. In case the ends
are sticky and there is a 4-base overhang, 16°C is optimum. In case of a 2-base
overhang and blunt ends, the optimum temperature is 12°C and 4°C respectively. This is because to anneal shorter fragments, lower temperature is required.
However, lower temperature reduces enzyme activity and enzyme concentration
should be adjusted as such.
T4 DNA ligase is much more efficient than E.Coli DNA ligase, especially
in blunt end ligation, and is preferred accordingly.
Most preparations of T4 DNA ligase are calibrated in Weiss units. A 0.015
Weiss unit of T4 DNA ligase will ligate 50% of Hind III fragments of bacteriophase
lambda (5/-tg) in 30 minutes at 16°C. Joining of blunt-ended molecule to one
another is improved greatly by the addition of noncovalent cation (150 to 200 mM
NaCl) and a low concentration of PEG.
Run aliquots of ligated DNA side by side with unligated DNA to minimize and
observe the difference. Being circular, ligated plasmid DNA will tend to run
faster than unligated plasmid DNA.