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SphI
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SphI Technical Brochure Technical Brochure
SphI is a restriction enzyme purified from Streptomyces phaeochromogenes.

 

Unit substrate: Lambda DNA.

 

Unit calculation assay conditions: 50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl2, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37oC.

 

Absence of contaminants: 50 units of SphI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA at 37oC. After 10-fold overdigestion with SphI, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.

 

Storage buffer: 100 mM KCl, 10 mM Tris-HCl  (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 400 μg/ml BSA and 50% glycerol. Store at -20oC.

 

Heat inactivation: 65oC for 20 minutes.

 

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

 

Percent Activity in MINOTECH Buffers
 L M  H  SH  A  K 
 75-100 100  50  50  50  100 
 
 
General reaction mixture:
10U SphI 1μl
10x M or K buffer*  2μl 
DNA substrate  <1μg
Sterile ultrapure water  Up to 20 μl
Incubate for 15 min at 37oC  
*In the case of M buffer we recommend the addition of BSA to a final concentration of 100 μg/ml. 

 

Reagents supplied: 10x M and 10x K buffer