The following protocol can be used to optimize ligation conditions for difficult to clone (e.g. very large) fragments. The principle is to independently characterize the ligation kinetics of the vector and insert DNA fragments and then to combine them in optimal ratios. The final ligation is also performed in two stages to optimize the proportion of vector:insert heterodimers followed by a shift to low concentration to optimize recircularization. Reactions are performed in sufficient quantities to permit analysis on agarose gels which allows reaction optimization, and identifies problematic substrates with unligatable ends or contamination with ligase inhibitors.

Materials:

T4 DNA ligase (and buffers from both Invitrogen and Roche).

0.1 M EDTA

3M NaOAc

70% EtOH

Glycogen 20 mg/mL (Roche)

TBE or TAE agarose gel

LB-Amp plates

Procedure:

1. Gel purify DNA restriction fragments of interest for both the vector and insert. Quantify the isolated fragments on a test gel with a DNA concentration standard. Optimize ligase concentration with fixed amount of Insert DNA:
2. Set up the first test ligation with serial dilutions of ligase to find the optimal enzyme concentration: Use 20 ng of insert DNA, 3 µL 5x Invitrogen ligation buffer, ligase and q.s. to 15 µL with water. Use 1 µL of enzyme in the first tube and then perform 10-fold serial dilutions of enzyme in 5 tubes. Incubate at room temperature for 15 minutes. Stop the reaction by adding 1/10 vol. of 0.1M EDTA and heat to 65°C for 5 min.
3. Run a TBE agarose test gel with 0.4 µg/mL ethidium bromide to resolve the ligated multimers. Use unligated DNA as a size control. Bands which run smaller than the unligated DNA are circular forms. Choose an enzyme concentration which gives maximal amount of dimers. If circular forms predominate then the DNA concentration was not high enough or there was no PEG in the ligase buffer. Optimize Vector DNA concentration:
4. Set up the second test ligation with serial dilutions of vector DNA to find the concentration which is optimal with the chosen enzyme concentration. Set it up as in step 2 except use a fixed concentration of ligase and instead titrate the vector DNA concentration (perform 2 or 4-fold dilutions). Run a test gel again and choose the DNA concentration which gives you optimal dimer formation. Alternatively, you can arbitrarily choose a vector DNA concentration which is 3-4 fold less (on a molar basis) than was used for the insert and skip to step 5. Ligate Vector and Insert to form a heterodimer:
5. Set up the final ligation as a two-step reaction: Use 20 ng of insert DNA, the optimal dilution of enzyme, and the optimized quantity of vector DNA. Ligate at room temperature for 15 minutes at room temperature. Remove and heat inactivate activate a 5 µL aliquot for a test gel. It is a good idea to also ligate vector only and insert only under the same condtions. Recircularize heterodimers:
6. Dilute the remainder of the ligation mix to a total volume of 250 µL with water, 2 µL of enzyme and 23 µL of 10x Roche ligation buffer (which does not contain PEG). Incubate at room temp. for 4 hours or overnight at 15°C.
7. Ethanol precipitate by adding 25 µL of 3M NaOAc, 1µL 20 mg/mL glycogen and 550 µL of ethanol at -20°C for 30 minutes. Spin for 10 minutes, and wash the pellet 2x with 70% ethanol. Resuspend in 5-10 µL of water and transform half of this into bacteria, and run the remainder on a test gel with the aliquots that were previously saved. On the test gel you should see both multimeric forms and circular forms. Occasionally, the addition of vector DNA to insert (or vice versa) will cause an inhibition of ligation. This is often due to contaminants from agarose gel purification. Agarose gel purification of vector DNA can often be avoided altogether by taking care to cut the vector DNA to completion (e.g. cut the DNA twice with phenol extraction and EtOH purification after each digestion).Because optimized ligation reactions may produce a large number of transformants it is important to perform 10-fold serial dilutions of the transformed bacteria while plating on to LB/Amp plates. If plasmid miniprep digests fail to identify the appropritate clone then plates the plates may be used for screening by colony hybridization.