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.
dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Impaired by overlapping
Activity in Simplebiotech buffers:
*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.
|DNA 0.5µg - 1µg||xµL|
|nuclease free water||up 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.