5'-G^A A T T C-3'
3'-C T T A A^G-5'
Supplied With:
R1625: Restriction Enzyme Buffer A, 10X
R1625-03: Restriction Enzyme Buffer D, 10X
Unit Definition: One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA in 1 hour at 37 degrees C in 50ul of assay buffer.
Incubation Temperature: 37 degrees C
Dilution:
For short-term storage (3-4 weeks):
Dilute with Diluent Buffer (R1625-05, 10mM Tris-HCl, pH 7.4 at 25 degrees C, 100mM potassium chloride, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.
For long-term storage:
Dilute with Storage Buffer (10mM potassium phosphate, pH 7.4, 25 degrees C, 300mM NaCl, 1mM EDTA, 7mM 2-mercaptoethanol, 0.2mg/ml BSA, 0.15% Triton X-100, 50% glycerol).
Quality Control:
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.
Ligation/Recutting Assay: After 50-fold overdigestion (3u/ug DNA x 17 hours) with EcoRI, more than 90% of the DNA fragments can be ligated at a 5'-termini concentration of 0.02uM. More than 90% of these can be recut.
Labeled Oligonucleotide Assay: No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10u of restriction endonuclease for 4 hours.
Blue/White Cloning Assay: pUC57 was digested at a unique site with 10u of enzyme for 16 hours. After religation and transformation 0.2% of white colonies were detected.
Methylation Effects: EcoRI cuts neither Gm6AATTC nor GAm6ATTC, but cuts GAATTm5C, GAATThm5C and GAAhm5Uhm5UC.
Storage and Stability:
Store at -20C. Stable for 12-12 months.
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