We have previously discussed restriction digestion, which constitutes the “cut” segment of the cloning process. With proper design, vector and insert DNA are engineered so that digestion with the same restriction enzyme(s) will produce compatible ends. These ends are complementary to each other and can be joined, or ligated together. Ligation is thus the “paste” step of the cloning process, and is achieved with the use of another class of enzymes: DNA ligases.
1. DNA ligases catalyze the formation of a phosphodiester bond between the 3′ hydroxyl terminus of one nucleotide and the 5′ phosphate terminus of another. Ligases are thus able to fix nicks in DNA, and play a critical role in DNA replication and repair in living organisms. In the lab, a specific enzyme, T4 DNA ligase, is used to join restriction enzyme products (vector and insert) having either blunt or cohesive ends, and form a recombinant DNA plasmid.
a. Before a ligation reaction is assembled, you need to know how much of each DNA counterpart to use, as well as the temperature and duration of the reaction.
- Vector-to-insert ratio: This ratio typically varies between 1:1 and 1:3 for cohesive ends. For blunt ends, a ratio of 1:5 is recommended, because ligation of blunt ends is a lot less efficient. I usually try several ratios (1:1, 1:2, and 1:3) and pick the one that gives the highest transformation efficiency (more on that in a later post). The ligation calculator from New England BioLabs is a nifty tool to calculate the mass of insert (in ng) required for several vector:insert ratios, based on the mass of the vector and the insert and vector lengths (in Kb).
- Incubation temperature and duration: An incubation at 16C (e.g. in a thermocyler) for 5-16 hours is very commonly used, especially for cohesive end ligation, and when high transformation efficiency is required (such as when constructing libraries). This temperature ensures a good balance between the activity of the ligase (optimal at 25C and diminished at low temperatures) and the annealing of the DNA ends. The latter is a lot more efficient at low temperatures, where it is easier for two DNA ends to bump into each other and stay together long enough to be joined by the enzyme. The annealing part of the ligation reaction can thus be made even more efficient at 4C, but at this temperature, the enzyme activity will be greatly reduced, and an overnight incubation will be required. Blunt-end ligations can be performed between 16C and 25C.
b. Termination of the reaction: T4 DNA ligase can be inactivated by incubation at 65C for 10-20 minutes. This step is usually recommended, except if using a quick ligation kit (which includes PEG). See the Tips section below.
c. Control reactions: As scientists, you know that control reactions are essential parts of any experimental procedure. They are very well worth the effort and can go a long way toward validating your results, as well as helping you troubleshoot. Every time I perform a ligation reaction (especially if working with a new vector), I run the following control reactions in parallel, using the same vector DNA concentration as the test reaction. The insert in these reactions is replaced with water.
- Undigested vector: You can think of this as a positive control reaction that checks the viability of the competent cells and the adequacy of the transformation procedure, and tests the antibiotic resistance of the plasmid. Uncut vector DNA will transform very efficiently into competent cells. If you get few colonies from this reaction, you should: (i) review your transformation protocol; (ii) prepare new antibiotic selection plates; and/or (iii) use a new batch of competent cells.
- Digested vector with ligase: A properly digested vector should not have compatible ends. If it does, the ligase will join these ends and the re-ligated vector will get efficiently transformed into the competent cells, and give rise to background colonies (i.e. colonies that harbor a vector without an insert).
- Digested vector without ligase: Any colonies obtained from this reaction are the result of uncut vector. This is most likely caused by inadequate preparation of the digested vector. Before assembling the ligation reaction, run digested vector alongside undigested vector on an agarose gel. You should see different migration patterns: the uncut supercoiled plasmid should appear to run faster, whereas the cut plasmid should run slower (higher on the gel). Also, keep in mind that digestion is not always 100% efficient, and some undigested vector may still linger in your digested prep. When gel-purifying your digested vector, run the prep on an agarose gel (0.6-0.8%) long enough to get a good separation between digested and undigested vector.
The number of colonies obtained from reactions 2 and 3 should be less than 5% (ideally less than 1%) of the number of colonies obtained from reaction 1.
d. Tips for successful ligation
- DNA gel purification of digested insert and vector DNA: DNA gels are commonly run with ethidium bromide, and the bands are visualized on a UV transilluminator. However, excessive exposure to UV light will damage the DNA and drastically reduce cloning efficiency. This effect can be mitigated by working quickly, using long-wave UV light, and limiting the exposure of DNA to the light. I personally prefer to use blue light (e.g. Safe Imager™ 2.0 Blue Light Transilluminator, Thermo Scientific), which is safer for me and my DNA preps, and is compatible with gel stains such as SYBR® Safe DNA (Thermo Scientific).
- ATP: The ligation reaction is dependent on ATP, an important component of the ligase buffer. If this buffer is older than one year or has been subjected to frequent freeze-thaw cycles, ATP may get degraded. It is thus advisable that the ligation buffer is divided into multiple small aliquots following the first use. ATP in the buffer may also precipitate upon storage, making it a good practice to vortex the ligase buffer vigorously prior to use.
- Salt: Ligation is inhibited by high salt concentrations. DNA preps should be cleaned (preferably gel-purified) prior to ligation.
- PEG: The ligase buffer included in quick ligation kits contains polyethylene glycol (PEG). Ligation reactions prepared with these kits should not be incubated overnight, nor heat-inactivated, as either will decrease the transformation efficiency. If transforming cells by electroporation (more on that in our next post), PEG must be removed from the ligation reaction using a DNA purification spin column.
Got more tips, advice, comments? Please share!
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