The problem may be that EcoRV leaves a blunt end so it compatible to ligate with anything else that approximates a blunt end. You probably would have much better results if the "center" enzyme site was a sticky end.

When performing a ligation reaction with one vector and two inserts, there are a few key considerations to increase the likelihood of successful ligation. Here are some recommendations:

1. Vector and Inserts Preparation: Ensure that the vector and inserts have been properly prepared and purified. This includes digesting the vector and inserts with the appropriate restriction enzymes, dephosphorylating the vector (if necessary) to prevent self-ligation, and purifying the DNA fragments from the digestion reaction using a DNA purification kit.

2. Insert Ratio Optimization: Determine the optimal molar ratio of vector to inserts. This ratio can vary depending on the specific experiment, but a standard recommendation is to use a two-fold molar excess of the vector compared to each insert. Adjust the amount of DNA accordingly to achieve the desired ratio.

3. Vector Control: Include a vector-only control in the ligation reaction. This control helps to identify any background self-ligation of the vector or potential contamination of the components.

4. Extended Incubation: Allow for a longer incubation time during ligation. Incubating the ligation reaction overnight at a lower temperature (such as 4°C) can improve the chances of successful ligation by giving the enzymes more time to bind DNA ends and create ligated products.

5. Optimal DNA Ligase Concentration: Determine and optimize the concentration of DNA ligase enzyme in the ligation reaction. It is recommended to follow the manufacturer's instructions or perform preliminary experiments to find the appropriate concentration that promotes efficient ligation without causing excess background ligation.

6. Ligation Enhancers: Consider using ligation enhancers, such as polyethylene glycol (PEG) or dimethyl sulfoxide (DMSO), to increase ligation efficiency. These additives can enhance DNA hybridization and enzyme activity, thus improving the likelihood of successful ligation.

7. Transformation and Selection: Transform the ligation reaction into competent host cells using an appropriate method (e.g., heat shock for bacterial cells). Following transformation, select transformed cells using selective media containing the appropriate antibiotic resistance marker present in the vector.

8. Verify and Validate: Perform colony screening, diagnostic PCR, or DNA sequencing to verify that the desired ligation product has been successfully obtained. This step is crucial to ensure that the correct inserts have been ligated into the vector.

By following these recommendations and optimizing the key parameters, you can increase the chances of obtaining successful ligation with one vector and two inserts. It is always advisable to consult relevant scientific literature or seek guidance from experienced researchers in your specific area of study to further refine your ligation strategy.