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Source
: Streptomyces phaechromogenes NRRL B-3559
Compatible ends
(CATG…3')
Bbu I, BstNS I, Hin1 II, Hsp92 II, Nla III, Nsp I, Pae I, SpaH I, Xce I
Isoschizomers
: Bbu I, Pae I, SpaH I
Methylation Sensitivity
: Not sensitive to dcm, dam and CpG methylation
Unit definition :
One Unit is defined as the amount of Sph I required to completely digest 1 ㎍ of λ DNA in one hour at 37℃ in 50 ㎕ assay mixture.
Typical Reaction Conditions
: Add 5 ㎕ of 10X Reaction Buffer 'Sph I'[final conc.: 50 mM Tris-HCl(pH 7.7 @ 37℃), 10 mM MgCl2, 100 mM KCl]], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Sph I and 42~43 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 37℃.
Heat Inactivation
: 65℃ for 20 min.
Activity in BeamsBio™ Buffer System
|
Rxn Buffer |
A |
B |
C★ |
D |
'Sph I' |
|
Activity(%) |
75~100 |
75~100 |
75~100 |
75~100 |
75~100 |
|
Activity in a complete PCR Mix : 75~100%
Conditions: [10 mM Tris-HCl(pH 8.3 @ 25℃), 50 mM KCl, 1.5 mM MgCl2, 2 pmol of primers, 200 μM dNTP Mix, 2.5 U Taq DNA polymerase and 1 ㎍ of DNA(template or substrate) in 50 ㎕ of reaction volume] After 30 amplication cycles, add 5 units of Sph I. And then incubate at 37℃ for one hour. Relative activity is determined by gel electrophoresis.
Storage Conditions
: 10 mM Tris-HCl(pH 7.5 @ 25℃), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.15% Triton™ X-100, 100 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.
No. of Cleavage Sites
|
λ
|
Ad2
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pUC18
|
φX174
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pBR322
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M13mp18
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SV40
|
|
6
|
8
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1
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0
|
1
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1
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2
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Sph I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Sph I for 16 hr in 50 ㎕ of reaction mixture at 37℃ as determined by agarose gel electrophoresis.
Ligation-Recut: After a 10-fold digestion for one hour, % of λ DNA fragments were ligated with T4 DNA ligase at 16℃ and % of the ligated fragments could be recut with Sph I.
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References
Fuchs, L.Y., Covarrubias, L., Escalante, L., Sanchez, S., Bolivar, F. Gene 10: 39-46 (1980)
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