Restriction digestion
One enzyme gives a linear vector with two identical ends, which can close on itself and can take an insert in either orientation. Two enzymes give non-identical ends, which fixes the orientation and largely stops self-closure, at the cost of needing a buffer and temperature where both are active.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Single / double digest
One enzyme gives a linear vector with two identical ends, which can close on itself and can take an insert in either orientation. Two enzymes give non-identical ends, which fixes the orientation and largely stops self-closure, at the cost of needing a buffer and temperature where both are active.
How it differs from the standard method
- A double digest sets the insert's orientation; a single digest does not, so clones must be screened for it.
- Both enzymes need acceptable activity in one buffer at one temperature, or you digest sequentially with a clean-up between.
- Keep the combined enzyme volume under about 5% of the reaction, or the glycerol alone can cause star activity.
- Always run uncut and single-digest lanes beside a double digest, or you cannot tell which of the two enzymes failed.
1A sequence-specific pair of scissors
A restriction endonuclease binds a short, usually palindromic sequence and cuts the double helix there. Most cloning enzymes cut within their recognition site and leave a short single-stranded overhang, a sticky end, which is why two fragments cut with the same enzyme can anneal to each other.
2How often a site occurs, and why that matters
A six-base site occurs by chance roughly once every 4,096 base pairs, so a 5 kb plasmid typically carries one or two sites for any given six-base enzyme and a human genome carries hundreds of thousands. That is the difference between an enzyme being a precise tool on a plasmid and a blunt instrument on genomic DNA.
3The reaction, and the three ways it goes wrong
Incomplete digestion
Not enough enzyme, too little time, or far more DNA than the units can handle. The gel shows the uncut band still present alongside the products, which looks exactly like having the wrong construct.
Star activity
Wrong salt, too much glycerol from a large enzyme volume, or an overnight digest with a big excess. The enzyme starts cutting sequences that merely resemble its site, giving extra bands and a smeared lane.
Methylation
Dam and Dcm methylation in E. coli blocks some enzymes at overlapping sites. The enzyme is fine and the conditions are fine; that particular site in DNA from that strain simply cannot be cut.
4Two enzymes in one tube
A double digest is convenient but adds constraints: both enzymes must be acceptably active in the same buffer and at the same temperature. Suppliers publish compatibility charts for exactly this reason.
5Why you dephosphorylate
A vector cut once has two compatible ends and will happily close on itself, which gives a plate full of colonies containing no insert. Treating it with a phosphatase removes the 5' phosphates, and since DNA ligase needs a 5' phosphate to seal a nick, the vector can no longer self-close.
Key terms
Practise these as flashcards →The short sequence an enzyme binds, usually 4–8 bp and often palindromic.
A short single-stranded overhang left by an offset cut; two ends from the same enzyme are complementary and anneal.
A cut straight across both strands, with no overhang. Compatible with any other blunt end, which makes it versatile but inefficient to ligate.
The amount of enzyme that digests 1 µg of a reference substrate in 1 hour under the supplier's conditions. It is a definition tied to that substrate, not a universal rate.
Cutting at sequences that only resemble the recognition site, caused by wrong salt, excess glycerol, excess enzyme or very long incubation.
Methylation added by E. coli that blocks some enzymes where it overlaps their site. A property of the host strain the DNA came from.
A digest chosen so that the fragment sizes distinguish the construct you want from the ones you might have got.
Removing 5' phosphates with a phosphatase so a singly cut vector cannot be re-sealed by ligase.
When to use it
- Cutting an insert out of one plasmid and opening another to receive it.
- Checking that a construct is correct, by predicting fragment sizes and comparing them with a gel.
- Distinguishing alleles when a variant creates or destroys a site, which is what RFLP genotyping does.
- Linearising a plasmid before in vitro transcription or before transfection.
Compared with alternatives
Prefer homology-based assembly when you want no scar, several fragments at once, or no suitable sites.
The step a digest usually feeds into; the quality of the digest sets the ceiling on the ligation.
How a digest is read: the sizes on the gel are the whole result.
RFLP uses a digest as the readout, with a band shift reporting the variant.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
One enzyme gives a linear vector with two identical ends, which can close on itself and can take an insert in either orientation. Two enzymes give non-identical ends, which fixes the orientation and largely stops self-closure, at the cost of needing a buffer and temperature where both are active.
- A double digest sets the insert's orientation; a single digest does not, so clones must be screened for it.
- Both enzymes need acceptable activity in one buffer at one temperature, or you digest sequentially with a clean-up between.
- Keep the combined enzyme volume under about 5% of the reaction, or the glycerol alone can cause star activity.
- Always run uncut and single-digest lanes beside a double digest, or you cannot tell which of the two enzymes failed.