Restriction endonucleases

Description

Product Details

Bcl I is a restriction enzyme purified from Bacillus caldolyticus.

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^–) in 60 min at 50^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 Bcl I	1μl
10x EQ buffer	5μl
DNA substrate	1μg
Sterile ultrapure water	Up to 50 μl

• Speed Digest reaction: Incubate for 15 minutes as in standard reaction in 1x R2 or 1x EQ

• Activity(%) in EnzyQuest buffers -Double digest tips

R1	R2	R3	R4	EQ
75	100	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: No

Methylation Sensitivity:

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

Unit calculation assay conditions:

• 1x R2 buffer
• Lambda DNA (dam^–)
• Incubation at 50^oC

Storage conditions:

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

Non-specific nuclease activity: 100 units of Bcl I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA (dam^–) at 50°C.

Re-ligation and recut: After 50-fold overdigestion with Bcl I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.