How restriction enzymes cut
Each enzyme recognizes a short sequence, often a palindrome of 4 to 8 bases, and cuts inside or next to it. EcoRI recognizes 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.
Predicting the fragments
Take a 4,000 bp plasmid with an EcoRI site at position 400 and a BamHI site at 1,300.
- Circular, cut with both: 1,300 − 400 = 900 bp and 4,000 − 900 = 3,100 bp.
- Circular, cut with EcoRI only: one cut opens the circle into a single 4,000 bp band.
- The same sequence as a linear molecule, cut with both: 400, 900 and 2,700 bp.
Uncut plasmid does not run at its true size on a gel: supercoiled DNA usually runs faster than the linear form, so always compare with a cut sample.
Common problems
- A site is there but the enzyme will not cut. DNA from common E. coli strains is methylated by Dam and Dcm, which blocks some enzymes where a site overlaps a methylated sequence. XbaI and ClaI are often affected. Grow the plasmid in a dam− dcm− strain if you need them.
- Extra bands appear. Too much enzyme, a long incubation or more than 5% glycerol in the reaction can make some enzymes cut at similar sequences, called star activity. Keep the enzyme volume to a tenth of the reaction or less.
- A site at the very end of a PCR product does not cut. Many enzymes need a few extra bases beyond the site to bind. Add 4 to 6 bases before the site in the primer.
- Two enzymes need different buffers. Check the supplier's double-digest table, or cut with one enzyme, clean up the DNA, then cut with the other.