Solving a restriction map The solution of a circular plasmid restriction map will include all restriction enzyme cutting sites for the given enzymes as well as the total length of the plasmid in kb and the distance in kb between each cut. 1. Only maps that were generated by restriction enzyme digestion with each single digest and all possible double digests can be unambiguously solved. Therefore, for three enzymes, one needs to perform at least six digests, three single and three double digests. 2. Make sure that the sum in kb of all the fragment lengths is identical in all six digests. The total pie must always be the same length. If in one digest the sum is too low, two bands may co-migrate because they are the same length. If in one the sum is too high, there may be extra bands due to partial digests. These bands will not be stoichiometric or their intensity will not be proportional to their fragment length. 3. Compare in each digest, the single versus double digest.
This is the end of the preview. Sign up
access the rest of the document.