|Molecular Biology||Storage: -20°CShipping: Blue Ice|
5'.....G A G^C T C.....3'
3'.....C T C^G A G.....5'
Unlike SacI, Ecl136II produces DNA fragments with blunt ends.
Source: Enterobacter cloacae RFL136
Concentration: 10 units/ul
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.
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).
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.
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.
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.
Enzyme is inactivated by incubation at 65°C for 20min.
Number of Recognition Sites in DNA: