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5'-G^A A T T C-3'
3'-C T T A A^G-5'
E.coli that carries the cloned ecoRIR gene from Escherichia coli RY13
R1625: Restriction Enzyme Buffer A, 10X
R1625-03: Restriction Enzyme Buffer D, 10X
(See E0375 for 10u/ul).
One unit is defined as the amount of EcoRI required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer.
Incubation Temperature: 37°C
For short-term storage (3-4 weeks):
Dilute with Diluent Buffer (R1625-05, 10mM Tris-HCl, pH 7.4 at 25°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°C, 300mM NaCl, 1mM EDTA, 7mM 2-mercaptoethanol, 0.2mg/ml BSA, 0.15% Triton X-100, 50% glycerol).
Stability during Prolonged Incubation:
A minimum of 0.2u of EcoRI is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Thermal Inactivation: EcoRI is inactivated by incubation at 65°C for 20min.
Digestion of Agarose-Embedded DNA:
A minimum of 5u of EcoRI is required for complete digestion of 1ug of agarose-embedded lambda DNA in 16 hours.
Compatible Ends: XapI, MunI, TasI
Number of Recognition Sites in DNA:
pBR322, pUC57, pUC18/19, pTZ19R/U,
Dam, Dcm, EcoKI, EcoBI: Never overlaps-no effect
CpG: May overlap-cleavage impaired
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 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.02uM. More than 95% 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 EcoRI for 4 hours.
Blue/White Cloning Assay: pUC57 was digested at a unique site with 10u of EcoRI for 16 hours. After religation and transformation 0.1% of white colonies were detected.
Protocol for Digestion:
Nuclease free water: 16ul
DNA (0.5-1ug/ml): 1ul
Mix gently and spin down for a few seconds. Incubate at 37ºC for 1-16 hours.
Protocol for Digestion Directly after Amplification:
PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA)
Nuclease free water: 18ul
Mix gently and spin down for a few seconds.
Incubate at 37ºC for 1-16 hours.