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5'-A G G^C C T-3'
3'-T C C^G G A-5'
Source: E.coli that carries the cloned eco147IR gene from E.coli RFL147
Restriction Enzyme Buffer A, 10X: (R1625)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.
Restriction Enzyme Buffer B, 10X: (R1625-01):
10mM Tris-HCl (pH 7.5), 10mM MgCl2 and 0.1mg/ml BSA. Incubate at 37°C
Storage Buffer: 10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
No detectable change in the specific fragmentation pattern is observed after 320-fold overdigestion (20u/ug lambda DNA x 16 hours) with Eco147I.
After 50-fold overdigestion (3u/ug DNA x 17 hours) with Eco147I, more than 90% of the DNA fragments can be ligated at a 5'-termini concentration of 0.03uM. More than 90% 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.
Blocked by overlapping Dcm methylation.
Blue/White Cloning Assay:
pUC57 was digested at a unique site with 10 units of enzyme for 16 hours. After religation and transformation 0.7% of white colonies were detected
Stability during Prolonged Incubation: A minimum of 0.1 units of enzyme is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Thermal Inactivation: Enzyme is inactivated by incubation at 80°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:
PhiX174, pUC57: 1
M13mp18/19, pBR322, pUC18/19, pTZ19R/U: 0