EcoR1 (EcoRI) Restriction Enzyme: Site, Cut and Buffer Activity

Short answer

EcoRI (often typed EcoR1 or “ecor 1”) is a Type II restriction endonuclease from Escherichia coli that cuts double-stranded DNA at the palindromic site 5′-GAATTC-3′, between G and A, leaving four-base 5′ AATT overhangs. The final character is the Roman numeral I, not the digit 1. This page gives the site and cut, the enzyme’s UniProt-recorded properties, and a buffer and star-activity reference for bench use.

What is EcoRI (EcoR1)?

EcoRI is a Type II restriction enzyme, so yes, it is a restriction enzyme: it recognises a fixed DNA sequence and cleaves within it. UniProt entry P00642 records it as R.EcoRI from Escherichia coli (TaxID 562), encoded on the plasmids pMB1 and pMB4, with the gene name ecoRIR. Promega lists the source strain as E. coli RY13. For the general classification of Type II enzymes, palindromic sites and isoschizomers, see restriction endonuclease types and isoschizomers.

Is it EcoRI or EcoR1? How is the name written?

The correct form is EcoRI. Search engines see EcoR1, ecor1, ecori and “ecor 1” as the same query, but catalogues, papers and UniProt use the Roman numeral I. The prefix R. (as in R.EcoRI) marks the restriction endonuclease as distinct from its cognate methyltransferase; the convention is explained in the restriction enzyme name index. Pronunciation is not standardised in the sources used here, so it is not given.

EcoRI recognition site and cut (GAATTC)

The EcoRI recognition site is 5′-GAATTC-3′. Per UniProt P00642 the enzyme cleaves after the G (position -1). Promega shows the same cut on the complementary strand, so the two strands are cut at different points:

5'-...G^AATTC...-3'
3'-...CTTAA^G...-5'

The result is a four-nucleotide 5′ overhang (AATT) on each fragment. Because every fragment end carries the same overhang, fragments from different EcoRI digests can be joined, which is why EcoRI sites appear in multiple cloning sites. UniProt notes the products carry 5′-phosphates.

Other names for the EcoRI site

“ecor1 restriction site”, “ecori recognition site” and “eco r 1 restriction site” all refer to the same hexanucleotide, GAATTC.

How EcoRI works

UniProt describes EcoRI as a homodimer that binds two Mg²⁺ ions per subunit; magnesium is the cofactor the cleavage reaction needs. The entry gives it the Enzyme Commission number EC 3.1.21.4. The structural facts below come from the same record.

Property	Value (UniProt P00642, read 2026-11-07)
Recognition sequence	5′-GAATTC-3′
Cut position	After G (-1); 5′ AATT overhang
Quaternary structure	Homodimer
Mature chain	277 amino acids (residues 2-277), 31,059 Da
Metal ions	Two Mg2+ per subunit
EC number	3.1.21.4
Example crystal structures	PDB 1ERI (2.50 Å), 1CL8 (1.80 Å)

EcoRI buffer activity and star activity

Promega’s product page for EcoRI (catalogue R6011, read 2026-11-07) recommends Buffer H at 37 °C. The activity it reports in each buffer is below. These percentages are for that product only; other suppliers’ buffers are formulated differently.

Buffer	Activity of EcoRI (Promega R6011)
Buffer A	25-50%
Buffer B, C or D	50-75%
Buffer H	100%
MULTI-CORE	100%, but not recommended because of star activity

Star activity risk factors

Star activity is cleavage at sequences similar to, but not exactly, the recognition site. The same page lists these conditions as able to cause it for EcoRI:

• glycerol above 12%
• more than 25 units of enzyme per µg of DNA
• no NaCl in the reaction
• Mn²⁺ present
• high pH

The practical reading is that the amount of enzyme and the glycerol it is supplied in add up: a large volume of a glycerol-containing stock can by itself push a small reaction past 12%. For how to assemble a digest, see restriction enzyme digest setup.

Where it matters for lab and supply decisions

Three practical choices follow from the Promega product page for EcoRI (read 2026-11-07).

• Buffer choice. The supplier-recommended buffer is Buffer H at 37 °C, where activity is reported as 100%. Buffer A gives 25-50% and Buffers B, C and D give 50-75%, so a digest planned in a different buffer needs more enzyme or time, or a change of buffer. MULTI-CORE is listed at 100% but carries a footnote that it is not recommended because of potential star activity.
• Star activity. The page lists glycerol above 12%, more than 25 units per µg of DNA, no NaCl, Mn²⁺ and high pH as conditions that can produce star activity. The storage buffer on the page contains 50% glycerol, so the volume of enzyme added to a reaction contributes to the final glycerol percentage.
• Standard versus high-concentration product. The page lists standard EcoRI (R6011, 5,000 units; R6017, 15,000 units) and a high-concentration product, EcoRI (HC), R4014 (25,000 units), at 40-80 U/µL. A higher concentration means a smaller volume per unit, which matters when comparing catalogue entries; check the concentration and pack size on the order line rather than assuming they match.

Catalogue numbers, pack sizes and buffers are supplier-specific and can change; confirm them against the current supplier page and the certificate for the lot in use.

Common misunderstandings

• “EcoR1” is a different enzyme. It is the same enzyme typed with a digit.
• The enzyme cuts straight through the site. It makes staggered cuts, leaving overhangs.
• 100% activity in any buffer. Activity depends on the buffer; see the table above.

Related concepts

• Other Type II restriction enzymes and isoschizomers. EcoRI is one member of a large family of enzymes that cut at defined sequences; the classification, and the idea of isoschizomers (different enzymes recognising the same site), are covered in restriction endonuclease types and isoschizomers.
• Enzyme naming. How names such as R.EcoRI are constructed is covered in the restriction enzyme name index.
• Setting up a digest. See restriction enzyme digest setup.
• Cloning vectors. EcoRI sites in multiple cloning sites.

FAQ

Is EcoRI a restriction enzyme?

Yes. It is a Type II restriction endonuclease (EC 3.1.21.4) that cleaves DNA at GAATTC.

What bacteria does EcoRI come from?

Escherichia coli. UniProt records the enzyme as encoded on plasmids pMB1 and pMB4 of E. coli, and Promega lists strain RY13 as the source.

How does EcoRI work?

As a homodimer it binds the GAATTC site, uses Mg²⁺ and cuts each strand between G and A, producing 5′ AATT overhangs.

What is the EcoRI recognition site sequence?

5′-GAATTC-3′, which reads GAATTC on both strands (a palindrome).

Sources

• UniProt P00642, Type II restriction enzyme EcoRI: rest.uniprot.org/uniprotkb/P00642.txt, read 2026-11-07.
• Promega, EcoRI (R6011) product page: promega.com EcoRI, read 2026-11-07.

This page is general reference for laboratory staff and is not a protocol; follow the supplier’s instructions for the lot in use.