EcoRI is a restriction enzyme purified from E. coli RY 13.
Reagents supplied: 10x EcoRI and 10x EQ buffer
Unit definition: One unit is the amount of enzyme required to digest 1 μg of Lambda DNA in 60 min at 37oC in a total reaction volume of 50 μl.
Standard reaction conditions: In order to overcome DNA source variability, a ~10 fold overdigest is suggested:
10U EcoR I
10x EQ buffer
Sterile ultrapure water
Up to 50 μl
Speed Digest reaction: Incubate for 15 minutes at 37oC as in standard reaction in 1x EcoRI buffer (50 mM NaCl, 100 mM Tris-HCl (pH 7.4 @ 25oC), 5 mM MgCl2, 0.025% Triton X-100, 100 μg/ml BSA) or 1x eQ buffer (20 mM Tris-acetate (pH 7.9 @ 25oC), 10 mM Mg-acetate, 50 mM K-acetate, 100 μg/ml BSA).
Activity(%) in EnzyQuest buffers-Double digest tips
Reactions were performed as for unit definition reaction. The above data could be used as guideline for double digest reactions. Our enzymes are also active and compatible in buffer systems from other vendors.
Heat inactivation: 65oC for 20 minutes
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG methylation: Impaired by overlapping
Unit calculation assay conditions:
1x EcoRI buffer
Incubation at 37oC
300 mM NaCl,
5 mM KPO4 (pH 7.4)
0.1 mM EDTA
1 mM dithiothreitol
0.15% Triton X-100
200 μg/ml BSA
Store at -20oC
Non-specific nuclease activity: 100 units of EcoR I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37oC.
Re-ligation and recut: After 50-fold overdigestion with EcoR I greater than 98% of the DNA fragments can be ligated and recut with this enzyme.
Star activity: Conditions of low ionic strength, high enzyme concentration, glycerol concentration >5%, or pH >8.0 may result in star activity.
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