SgrBI is a restriction enzyme purified from Streptomyces griseus.
Reagents supplied: 10x SgrBI and 10x EQ buffer
Unit definition: One unit is the amount of enzyme required to digest 1 μg of Lambda DNA (Hind III digest) in 60 min at 37oC in a total reaction volume of 50 μl.
Standard reaction conditions: In order to overcome DNA source variability, a ~10 fold overdigest is suggested:
10U SgrB I
10x EQ buffer
Sterile ultrapure water
Up to 50 μl
Speed Digest reaction: Incubate for 15 minutes at 37oC as in standard reaction in 1x SgrBI buffer (10 mM Tris-HCl (pH 7.9@ 25°C), 10 mM MgCl2, 0.1% Triton X-100, 100 μg/ml BSA)or 1x eQ buffer (20 mM Tris-acetate (pH 7.9 @ 25oC), 10 mM Mg-acetate, 50 mM K-acetate, 100 μg/ml BSA).
Activity(%) in EnzyQuest buffers-Double digest tips
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
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG methylation: Blocked
Unit calculation assay conditions:
1x SgrBI buffer
Lambda DNA (Hind III digest)
Incubation at 37oC
50 mM NaCl,
10 mM Tris-HCl (pH 7.4)
0.1 mM EDTA
1 mM dithiothreitol
200 μg/ml BSA
Store at -20oC
Non-specific nuclease activity: 400 units of SgrB I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA (Hind III digest) at 37°C.
Re-ligation and recut: After 100-fold overdigestion with SgrB I, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.
Note: Particular sites in λ and φΧ174 DNAs are difficult to cleave with SgrB I, as well as with its prototype Sac II.
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