EcoRI
(Escherichia coli RY 13)
5’-G A A T T C-3’
3’-C T T A A G-5’
Reaction Temperature: 37°C
Prototype: EcoRI
Inactivation Temperature (20 min): 65°C
Reaction Buffer:
1x High Buffer: 50 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl, 1 mM ditiothreitol, 0,025% Triton X-100, 100 µg/ml bovine serum albumin.
Unit Definition: One unit is the amount of enzyme required to completely digest 1 µg of lambda DNA in 1 hr in a total reaction volume of 50 µl.
Assay Conditions: 50 mM Tris-HCl (pH 7.5 at 37°C), 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 0,025% Triton X-100, 1 µg of lambda DNA and 100 µg/ml bovine serum albumin. Incubation is at 37°C for 1 hr in a reaction volume of 50 µl.
Storage Buffer: 10 mM potassium phosphate (pH 7.5), 300 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.2% (v/v) Triton™X-100, 100 µg/ml bovine serum albumin, 50% (v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Non-specific Endonuclease: Incubation of 30 units of EcoRI with 1 µg of lambda DNA at 37°C for 16 hrs (a 480-fold over-digestion) resulted in the same sharp characteristic banding pattern as the standard assay reaction, as determined by agarose gel electrophoresis.
3'-Exonuclease: 5, 10 and 20 units of EcoRI and 0.08 µg of 3'-ends of lambda/TaqI fragments (3'-labeled with T4 DNA Polymerase and [³H]dGTP and [³H]dCTP), incubated for 1 hr at 37°C resulted in 0.01-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
5'-Exonuclease/5'-Phosphatase: Incubation of 5, 10 and 20 units of EcoRI with 0.013 µg pmol of 5'-ends of [5'-³²P]lambda/HaeIII fragments for 1 hr at 37°C resulted in 0.09-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.