Restriction endonucleases

Description

Product Details

BstE II is a restriction enzyme purified from Bacillus stearothermophilus.

Reagents supplied: 10x BstEII and 10x EQ buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of Lambda DNA in 60 min at 60^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 BstE II	1μl
10x EQ buffer	5μl
DNA substrate	1μg
Sterile ultrapure water	Up to 50 μl

• Speed Digest reaction: Incubate for 15 minutes at 60^oC as in standard reaction in 1x BstEII buffer (100 mM KCl, 10 mM Tris-HCl (pH 7.4 @ 25°C), 5 mM MgCl2, 0.1% Triton X-100, 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
50	50-75	75-100	50	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: No

Methylation Sensitivity:

• dam methylation: Not sensitive
• dcm methylation: Not sensitive
• CpG methylation: Not sensitive

Unit calculation assay conditions:

• 1x BstEII buffer
• Lambda DNA
• Incubation at 60^oC

Storage conditions:

• 100 mM KCl,
• 10 mM Tris-HCl (pH 7.4)
• 5 mM MgCl₂
• 0.1 mM EDTA
• 1 mM dithiothreitol
• 200 μg/ml BSA
• 50% glycerol
• Store at -20^oC

Non-specific nuclease activity: 150 units of BstE II do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 60^oC.

Re-ligation and recut: After 100-fold overdigestion with BstE II, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Note: BstE II exhibits 10-15% activity at 37°C.