Restriction endonucleases

Description

Product Details

XbaI is a restriction enzyme purified from Xanthomonas badrii.

Reagents supplied: 10x R2 and 10x eQ buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of Lambda DNA (dam^–/Hind III digest) in 60 min at 37^oC in a total reaction volume of 50 μl.

Digest Guidelines 

• Standard reaction conditions: In order to overcome DNA source variability, a ~10 fold overdigest is suggested: 

10U Xba I	1μl
10x eQ buffer	5μl
DNA substrate	1μg
Sterile ultrapure water	Up to 50 μl
Incubate for 60 min at 37ºC

• Speed Digest reaction: Incubate for 15 minutes at 37^oC as in standard reaction in 1x R2 buffer (50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25^oC), 10 mM MgCl2, 100 μg/ml BSA) or 1x eQ buffer (20 mM Tris-acetate (pH 7.9 @ 25^oC), 10 mM Mg-acetate, 50 mM K-acetate, 100 μg/ml BSA).
• Activity(%) in EnzyQuest buffers -Double digest tips 

R1	R2	R3	R4	eQ
10-25	50-75	75-100	50-75	100

Reactions were performed as for unit definition reaction. The above data could be used as guideline for double digest reactions. Our enzymes are also active and compatible in buffer systems from other vendors.   

Heat inactivation: 65ºC for 20 minutes 

Methylation Sensitivity: 

• dam methylation: Blocked by overlapping 
• dcm methylation: Not sensitive 
• CpG methylation: Not sensitive 

 Unit calculation assay conditions:  

• 1x R2 buffer 
• Lambda DNA (dam-/Hind III digest) 
• Incubation at 37ºC 

 Storage conditions:  

• 100 mM KCl,  
• 10 mM Tris-HCl (pH 7.4) 
• 0.1 mM EDTA  
• 1 mM dithiothreitol 
• 500 μg/ml BSA 
• 50% glycerol  
• Store at -20ºC

Non-specific nuclease activity: 200 units of Xba I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA (dam–/Hind III digest)at 37ºC  

Re-ligation and recut: After 100-fold overdigestion with Xba I, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.