Once you have loaded two sets of sequences into the program you can create an alignment. The alignments in re-DOT-able are word-based, meaning that it looks for exact matches of a given length or longer.
There are two values which determine the alignment parameters within re-DOT-able. The main one is the search word size. This is the minimum match length which will be reported by the program. You can interactively increase this cutoff in the interactive view, but the value set when running the alignment is the lower bound, which can't be changed without recalculating the alignment.
The default word size for re-DOT-able is 50, but this can be lowered by going to View > Preferences. Any change in setting will take effect when the next alignment is calculated. Making the value lower will report shorter matches, and will result in a more sensitive (but possibly slower and noisier) search.
The other value which relates to the alignment is the index word size. The search algorithm operates by hashing the k-mer positions in one of the sequences and looking them up in the other. The index word size defines the kmer size used for the hashing, and therefore also defines the minimum word size which can be used for the search. Currently re-DOT-able uses an index word size of 10, and this isn't currently configurable.
Once you have set the word size you want to use for your search, you can start the alignment using File > Start Aligning.