| 5-A G C T-3 3-T C G A-5 Source: Arthrobacter luteus Concentration: 10u/ul Supplied With: R1625 - Restriction Enzyme Buffer A, 10X: Dilute to 1X for use. 1X buffer composition is 33mM Tris-acetate (pH 7.9 at 37C), 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA Diluent Buffer: 10mM Tris-HCl (pH 7.4), 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol Unit Definition: One unit is defined as the amount of Alul required to digest 1ug of lambda DNA in 1 hour at 37C in 50ul of assay buffer. Stability during Prolonged Incubation: A minimum of 0.1units of enzyme is required for complete digestion of 1ug of lambda DNA in 16 hours at 37C. Thermal Inactivation: Enzyme is inactivated by incubation at 65C for 20 minutes Methylation Effects: Dam, Dcm, CpG,EcoKl: never overlaps - no effect. EcoBl: may overlap - blocked Number of Recognition Sites in DNA: Lambda: 143 PhiX174: 24 pBR322: 1 pUC5: 16 pUC18/19: 16 pTZ19R/U: 1 M13mp18/19: 27 Overdigestion Assay: No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with AluI. Ligation/Recleavage Assay: The ligation and recleavage assay was replaced with LO test after validating experiments showed LO test ability to trace nuclease and phosphatase activities with sensitivity that is higher than L/R by a factor of 100. Labeled Oligonucleotide (LO) Assay: No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10units of restriction endonuclease for 4 hours. Protocol for Digestion: Add: Nuclease free water: 16ul R1625: 2ul DNA (0.5-1ug/ml): 1ul A1365: 0.5-2ul Mix gently and spin down for a few seconds. Incubate at 37C 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: 2ul A1365: 1-2ul Mix gently and spin down for a few seconds. Incubate at 37C for 1-16 hours. |
