Use of controls in USER cloning

As in any other scientific experiment, the use of controls is needed to confirm that the results are due to biological variables and not contamination or issues in the method. In the case of USER, it is important to have at least a negative control to check the re-ligation efficiency of the vector that is being assembled.

By doing this, one can get an estimation of the ability of the original vector to re-ligate and generate colonies. For instance, getting 1000 colonies after transforming bacteria with the sample, and getting 300 in the negative control, indicates that only 70% of the colonies will contain the assembled vector. In order for the researcher to know which colonies are the ones they need, they need to extract and sequence the DNA of several colonies to confirm those targeted.

However, there could be some exceptions to the addition of negative controls when performing a USER cloning experiment. If the outcome of the cloning is a given phenotype, for instance, the production of a colored compound, researchers would know the re-ligation efficiency by counting the number of colored colonies vs the number of uncolored ones. Nevertheless, the use of negative controls is always highly recommended.