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5'-G^A A T T C-3'
3'-C T T A A^G-5'
E.coli that carries the cloned ecoRIR gene from Escherichia coli RY13
R1625: Restriction Enzyme Buffer A, 10X: Supplied as a liquid in 33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA (pH 7.9 at 37°C).
R1625-03: Restriction Enzyme Buffer D, 10X: Supplied in 50mM Tris-HCl, pH 7.5, 10mM MgCl2, 100mM NaCl, 0.02% Triton X-100, 0.1mg/ml BSA.
(See E0375-05 for 50u/ul).
One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer.
Incubation Temperature: 37°C
10mM Tris-HCl, pH 7.4 at 25°C, 100mM potassium chloride, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.
Supplied in 10mM potassium phosphate, pH 7.4, 25°C, 300mM NaCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 0.15% Triton X-100, 50% glycerol.
Stability during Prolonged Incubation: A minimum of 0.2u of EcoRI are required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Thermal Inactivation: EcoRI is inactivated by incubation at 65°C for 20min.
Digestion of Agarose-Embedded DNA: A minimum of 5u of EcoRI is required for complete digestion of 1ug of agarose-embedded lambda DNA in 16 hours.
Compatible Ends: XapI, MunI, TasI
Number of Recognition Sites in DNA:
Overdigestion Assay: No detectable change in the specific fragmentation pattern is observed after 80-fold overdigestion (5u/ug lambda DNA x 16 hours) with EcoRI.
The ligation and recleavage assay was replaced with LO test after validating experiments showed LO test ability to trace nuclease and phosphatase activities with sensitivity that is higher than L/R by a factor of 100.
Labeled Oligonucleotide Assay: No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10u of EcoRI for 4 hours.
Blue/White Cloning Assay: The B/W assay was replaced with LO test after validating experiments showed LO test ability to detect nuclease and phosphatase activities with sensitivity that equals to that of B/W test.
Dam, Dcm, EcoBl, EcoKl: never overlaps - no effect,
CpG: may overlap - cleavage impaired.