5'-C A C N N N^G T G-3'|3'-G T G^N N N C A C-5' ||Source: |Alcaligenes denitrificans SS 3–028||Concentration: |10u/ul||Incubation Temperature: |37°C||Diluent Buffer: |10mM Tris-HCl pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol||Storage Buffer: |Supplied as a liquid in 10mM Tris-HCl pH 7.5, 300mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol||Supplied with:|R1625: Restriction Enzyme Buffer A, 10X: Dilute to 1X for use. 1X buffer composition is 33mM Tris-acetate pH 7.9 at 37°C, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA.||R1625-02: Restriction Enzyme Buffer C, 10X, Dilute to 1X for use. 1X buffer composition is 10mM Tris-HCl pH 7.5 10mM magnesium chloride, 50mM sodium chloride, 0.1mg/ml BSA.| |Unit Definition: |One unit is defined as the amount of AdeI required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer.||Enzyme Properties:|Star Activity: |An excess of AdeI (20u/ug DNA x 1 hour) may result in star activity.||Methylation Effects:|Dam, Dcm: Never overlaps - No effect.|CpG: May overlap - cleavage impaired.|EcoKl, EcoBl: May overlap - Effect not determined.||Stability during Prolonged Incubation: |A minimum of 0.5units of AdeI is required for complete digestion of 1ug of DNA in 16 hours at 37°C. ||Digestion of Agarose-embedded DNA: |A minimum of 10units of AdeI is required for complete digestion of 1ug of agarose-embedded lambda DNA in 16 hours. | |Number of Recognition Sites in DNA: |Lambda: 10 |PhiX174, pTZ19R/U, M13mp18/19: 1|pBR322, pUC18/19, pUC57: 0||Thermal Inactivation: |AdeI is not inactivated by incubation at 80°C for 20min (see Protocol for Inactivation Procedure.||Quality Control:|Overdigestion Assay: |No detectable change in the specific fragmentation pattern is observed after 15-fold overdigestion (15u/ug lambda DNA x 1hour) with AdeI (see Star Activity).||Ligation and Recleavage (L/R) Assay: |After a 10-fold overdigestion (5u/ug DNA x 2 hours) with Adel, more than 80% of the digested DNA fragments can be ligated at a 5’-termini concentration of 0.1uM. More than 90% of these sites can be recut.||Labeled Oligonucleotide (LO) Assay: |No detectable degradation of a single-stranded or double-stranded labeled oligonucleotide occurred during incubation with 10 units of AdeI for 4 hours. ||Blue/White (B/W) Cloning Assay: |A mixture of pUC57/Hindlll, pUC57/Eco32l and pUC57/Pstl digests when incubated with 10 units of Adel for 1 hour. After religation and transformation, the background level of white colonies was <1%.||Storage and Stability:|May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months after receipt at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.