| 5'-G^A A T T C-3' |
|
| 3'-C T T A A^G-5' |
|
| Catalog # | E0375-05 |
| Source | E.coli that carries the cloned ecoRIR gene from Escherichia coli RY13 |
| Supplied With | R1625: Restriction Enzyme Buffer A, 10X |
| R1625-03: Restriction Enzyme Buffer D, 10X |
| Concentration | 50u/ul. |
| (See E0375 for 10u/ul). |
| Unit Definition | 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 |
| Dilution | 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). |
| Enzyme Properties | 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: |
| Lambda: 5 |
| PhiX174: 0 |
| pBR322, pUC57, pUC18/19, pTZ19R/U, |
| M13mp18/19: 1 |
| Methylation Effects: |
| Dam, Dcm, EcoKI, EcoBI: Never overlaps-no effect |
| CpG: May overlap-cleavage impaired |
| 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 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.02uM. More than 95% of these can be recut. |
| Quality Control | |
| 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 | |
| Add | |
| Nuclease free water | 16ul |
| R1625-03 | 2ul |
| DNA (0.5-1ug/ml) | 1ul |
| E0375-05 | 0.5-2ul |
| Mix gently and spin down for a few seconds. Incubate at 37ºC for 1-16 hours. |
|
| Protocol for Digestion Directly after Amplification | |
| Add | |
| PCR Reaction Mixture | 10ul (~0.1-0.5ug of DNA) |
| Nuclease free water | 18ul |
| R1625-03 | 2ul |
| E0375-05 | 1-2ul |
| Mix gently and spin down for a few seconds. |
| Incubate at 37ºC for 1-16 hours. |
