5'-Pu^A A T T Py-3' 3'-Py T T A A^Pu-5'
Source
Xanthomonas ampelina Slo 51–021
Form
Supplied as a liquid (Storage Buffer) in 10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
Restriction Enzyme Buffer A, 10X (for 100% digestion) R1625
33mM Tris-acetate, pH 7.9, 10mM magnesium acetate, 66mM potassium acetate and 0.1mg/ml BSA. Incubate at 37°C.
Unit Definition
One unit is defined as the amount of XapI required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer.
Overdigestion Assay
No detectable change in the specific fragmentation pattern is observed after 15-fold overdigestion with XapI(15u/ug lambda DNA x 1 hours) .
Ligation / Recutting Assay
After 10-fold overdigestion (5u/ug DNA x 2 hours) with HpaII, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.2uM. More than 95% of these can be recut.
Labeled Oligonucleotide (LO) Assay
No detectable degradation of single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of XapI for 4 hours.
Blue/White Cloning Assay
pUC57 was digested at a unique site with 10 units of XapI for 1 hours. After religation and transformation 0.2 % of white colonies were detected.
Star Activity
An excess of XapI (20u/ug DNA x 1 hour) may result in star activity.
Stability during Prolonged Incubation
A minimum of 0.1 units of XapI is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Digestion of Agarose-embedded DNA
A minimum of 5 units of XapI is required for digestion of 1ug of agarose-embedded lambda DNA in 16 hours.
Compatible Ends
EcoRI, MunI, TasI
Number of Recognition Sites in DNA
Lambda: 58PhiX174: 5 pBR322: 1 pUC57: 1 pUC18/19: 1 pTZ19R/U: 3 M13mp18/19: 11
Protocol for Digestion
Add: 44ul nudlease free dH2O 5ul Restriction Enzyme Buffer A, 10X 1ul DNA (0.5-1ug/ul) 0.5-2ul XapI Mix gently and spin down for a few seconds. Incubate at 3ºC for 1-2 hours.
Protocol for Digestion of PCR products Directly after Amplification
Add: 10ul (~1ug DNA) 16ul nuclease free dH2O 2ul Restriction Enzyme Buffer A, 10X 1-2ul XapI Mix gently and spin down for a few seconds. Incubate at 3ºC for 1-16 hours.
Thermal Inactivation
XapI is inactivated by incubation at 65°C for 20min.
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. Further dilutions can be made in assay buffer.