More than 80% of the digested DNA fragments can be ligated and more than 95% can be recut in a reaction mixture containing 20 to 40U of T4 DNA ligase/1μg of fragments and 10% PEG at a 5ft.-termini concentration of 0.13μM after a 50-fold overdigestion(3U/μg DNA x 17 hours) with BfuI FspBI (BfaI).
5'...C▵T A G...3'
3'...G A T▵C...5'
Conditions for 100% Activity:
1X Buffer Tango™ :33mM Tris-acetate (pH7.9 at 37°C), 10mM Mg-acetate, 66mM K-acetate and 0.1mg/mL BSA
Incubate at 37°C
FspBI is supplied in: 10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/mL BSA and 50% (v/v) glycerol
Dam: may overlap — no effect
Dcm: never overlaps — no effect
CpG: never overlaps — no effect
EcoKI: never overlaps — no effect
EcoBI: never overlaps — no effect
Digestion of Agarose-embedded DNA:
Minimum 10U of the enzyme are required for complete digestion of 1μg of agarose-embedded lambda DNA in 4 hours
FspBI cleavage is impaired by overlappingdam methylation. To avoiddam methylation, use adam-, dcm strain such as GM2163 (#M0099).