How restriction enzymes cut
Each enzyme recognises a short sequence, often a palindrome of 4 to 8 bases, and cuts inside or next to it. EcoRI recognises GAATTC and cuts between G and A on both strands, leaving four-base AATT overhangs, called sticky ends. EcoRV cuts GATATC straight through the middle, leaving blunt ends.
Find which enzymes cut
- Paste the sequence into the Restriction Summary.
- Choose linear or circular. A plasmid is circular.
- Read the list of enzymes, the number of cuts, the positions and the fragment sizes.
Enzymes that cut once are the ones to use for opening a plasmid. On a circle, n cuts give n fragments; on a linear molecule they give n + 1.
See the sites on the sequence
The Restriction Map prints the sequence with every cut site marked above it, which makes it easy to check that a site sits where you think it does, or to find two enzymes either side of a gene.
Add a site without changing the protein
If there is no convenient site, one or two silent mutations can sometimes create one while the protein stays the same. Mutate for Digest lists those places in a coding sequence, ready for site-directed mutagenesis or to design into a primer.