Abstract
Differential digests, also known as test or diagnostic digests, are a standard method in molecular cloning to verify whether a picked clone is indeed the target plasmid or not. However, finding the optimal restriction enzyme for a differential digest by hand may be challenging and time-consuming. To address this problem, we created diffdigester.uni-jena.de (https://diffdigester.uni-jena.de), a free online tool to easily find such enzymes. This tool uses regular expressions to find the restriction sites in the DNA sequences given by the user. It then calculates and displays the resulting fragments on a simulated gel for each enzyme, allowing for easy comparison between the different enzymes. The user can sort these gels alphabetically by enzyme name or by dissimilarity of the restriction patterns between the given DNA sequences. This way, the most distinct pattern is shown first, which gives the most useful enzyme to distinguish between wanted and unwanted ligation products. In fact, it also works on completely unrelated sequences, expanding its possible applications. Thus, diffdigester.uni-jena.de is a fast, reliable, and free-to-use tool to help researchers plan differential digests for verifying their ligation products.