| 5'.....G A G^C T C.....3' |
|
| 3'.....C T C^G A G.....5' |
|
| Catalog # | E0274 |
| Unlike SacI, Ecl136II produces DNA fragments with blunt ends. |
| Source | Enterobacter cloacae RFL136 |
| Concentration | 10 units/ul |
| Unit Definition | One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA-HindIII fragments in 1 hour at 37°C in 50ul of assay buffer. |
| Supplied with | R1625: Restriction Enzyme Buffer A, 10X: Supplied as a liquid in 33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA (pH 7.9 at 37ºC). |
| Supplied with | R1625-55: Restriction Enzyme Buffer for Ecl136II, 10X: Supplied as a liquid 10mM Bis-Tris Propane-HCl, pH 6.5, 10mM MgCl2, 0.1mg/ml BSA. Incubate at 37°C. |
| Diluent and Storage Buffer | 10mM Tris-HCl , pH 7.4 at 25°C, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol. |
| Quality Control | |
| Overdigestion Assay | |
| No detectable change in the specific fragmentation pattern is observed after 320-fold overdigestion (20u/ug lambda DNA x 16 hours) with Ecl136II. |
|
| Ligation/Recutting Assay | |
| After 50-fold overdigestion (3u/ug DNA x 17 hours) with Ecl136II, more than 90% of the DNA fragments can be ligated at a 5'-termini concentration of 0.01uM. 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. |
|
| Blue/White Cloning Assay | |
| pUC57 was digested at a unique site within lacZ gene with 10 units of enzyme for 16 hours. After religation and transformation 1% of white colonies were detected. |
|
| Methylation Effects | |
| The hemi-methylated sequence GAGCTm5C is cleaved by Ecl136II at a significantly slower rate. Cleavage is blocked when both strands are methylated at positions | 5'… GAGCTm5C…3' 3'…m5CTCGA G…5' [Overlapping CG methylation may influence DNA cleavage] |
|
| Stability during Prolonged Incubation | |
| A minimum of 0.2 units of enzyme are required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C. |
|
| Thermal Inactivation | |
| Enzyme is inactivated by incubation at 65°C for 20min. |
|
| Number of Recognition Sites in DNA | |
| Lambda 2 |
| PhiX174 0 |
| M13mp18/19 1 |
| pBR322 0 |
| pUC18/19 1 |
| pUC57 1 |
| pTZ19R/U 1 |
|
