Restriction endonucleases

Description

Product Details

SfiI is a restriction enzyme purified from Streptomyces fimbriatus (ATCC 15051).

Reagents supplied: 10x R2 and 10x EQ buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of Adenovirus-2 DNA 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 Sfi I	1μl
10x EQ buffer	5μl
DNA substrate	1μg
Sterile ultrapure water	Up to 50 μl

• Speed Digest reaction: Incubate for 15 minutes at 50^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
75-100	100	25-50	10-25	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: Impaired by overlapping
• CpG methylation: Blocked by some combinations of overlapping

Unit calculation assay conditions:

• 1x R2 buffer
• Adenovirus-2 DNA
• Incubation at 37^oC

Storage conditions:

• 300 mM NaCl,
• 5 mM KPO₄ (pH 7.4)
• 0.1 mM EDTA
• 1 mM dithiothreitol
• 0.15% Triton X-100
• 500 μg/ml BSA
• 50% glycerol
• Store at -20^oC

Non-specific nuclease activity: 100 units of Sfi I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Adeno-2 DNA at 50^oC.

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