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EcoRI is a restriction enzyme isolated from E. coli RY 13 that cleaves the palindromic sequence G▼AATTC.

Concentration: 10U/µL
Source: Escherichia coli RY13
Reagents supplied with: The Enzyme is supplied with 10x UniqueEcoRI Buffer (recommended) and the universal Simple Buffer for double digests. The enzyme is also fully active in the respective buffers from other suppliers.

Store at: -20 °C

Star activity: Conditions of low ionic strength, high enzyme concentration, glycerol concentration or pH>8.0 may result in star activity.


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EcoRI Restriction Endonuclease

Unit definition: One unit is defined as amount of enzyme required to produce a complete digest of 1 µg Lambda DNA in 60 min in a total reaction volume of 50µL under the assay conditions.
Unit substrate: Lambda DNA.
Unit calculation assay conditions: 1 µg Lambda DNA has been digested in 100 mM Tris-HCl (pH 7.4 at 25 °C), 50mM NaCl, 5mM MgCl2, 0.025% Triton X-100 ,100 µg/mL BSA
Absence of contaminants: 50 units of EcoRI do not produce any unspecific cleavage products after 16 hrs incubation with 1µg of Lambda DNA at 37 °C. After 50-fold overdigestion with EcoRI, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.
Storage buffer: EcoRI is supplied in 300 mM NaCl, 5 mM KPO4 (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 0.15% Triton, 200 μg/ml BSA and 50% glycerol.
Inactivation: Heat to 65 °C for 20 minutes. It is also possible to stop the digest by addition of EDTA to a final concentration of >10mM or SDS to a concentration of 0.1 %. Alternatively use our environmentally friendly 6x orange gel loading buffer. For further enzymatic reactions remove SDS or EDTA by spin column-based PCR purification kit.
Star activity: Conditions of low ionic strength, high enzyme concentration, glycerol concentration>5%, or pH>8.0 may result in star activity.
Methylation Sensitivity:
dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Impaired by overlapping

Activity in Simplebiotech buffers:

L M H SH Simple 2x Simple
25-50* 50-75 75 50-75 100 100

*May exhibit star activity under these conditions.

10x UniqueEcoRI buffer: 1000 mM Tris-HCl (pH 7.4 at 25 °C), 500mM NaCl, 50mM MgCl2, 0.25% Triton X-100 ,1mg/mL BSA
10x Simple buffer: 500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, 1000 μg/ml BSA, pH 7.9 at 25°C

Rapid digest within 15 minutes.

ComponentFinal concentration
Buffer UEcoRI2µL
DNA 0.5µg - 1µgxµL
nuclease free waterup to 20µL

Incubate at 37 °C for 15 minutes.
Always add the restriction enzymes as last component. Mix gently but thoroughly by pipetting up and down or flicking. Spin down.
Incubate at 37 °C for 15min. Restriction enzymes are stored in 50% glycerol. The glycerol concentration in the reaction mix should not exceed 10%. When digesting more than 1µg DNA, scale up the restriction mix or prolong the incubation time.


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