|Molecular Biology||Storage: -20°CShipping: Blue Ice|
5'-G G T C T C(N)1^-3'
3'-C C A G A G(N)5^-5'
E. coli that carries the cloned eco31IR gene from E. coli RFL31
Reaction Buffer (10X) (E0373-55A):
Tris-HCl (pH 7.5), 10mM MgCl2, 50mM NaCl and 0.1mg/ml BSA. Incubate at 37°C
Restriction Enzyme Buffer A, 10X:
33mM Tris-acetate (pH 7.9), 10mM magnesium acetate, 66mM potassium acetate and 0.1mg/ml BSA. Incubate at 37°C. Supplied to simplify buffer selection for double digests.
10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol, or Storage Buffer.
10mM Tris-HCl (pH 7.5 at 25°C), 200mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
No detectable change in the specific fragmentation pattern is observed after 80-fold overdigestion (5u/ug lambda DNA x 16 hours) with Eco31I (see Star Activity).
After 50-fold overdigestion (3u/ug DNA x 17 hours) with Eco31I, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.05uM. More than 95% of these can be recut.
Labeled Oligonucleotide (LO)Assay:
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of restriction endonuclease for 4 hours.
A large excess of enzyme (10u/ug DNA x 16 hours) may result in star activity.
Sequence GAGACm5C is cleaved by Eco31I at least 50-fold slower than an unmethylated site. Overlapping Dcm or CG methylation may influence DNA cleavage.
Stability during Prolonged Incubation:
A minimum of 0.3 units of enzyme is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Enzyme is inactivated by incubation at 65°C for 20min.
Digestion of Agarose-embedded DNA:
A minimum of 5 units of enzyme is required for digestion of 1ug of agarose-embedded lambda DNA in 16 hours.
Number of Recognition Sites in DNA:
One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA dcm--HindIII fragments in 1 hour at 37°C in 50ul of assay buffer. Assayed using lambda DNA dcm-, as one of two Eco31I recognition sites is difficult to cleave.
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.