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Source
: Sphaerotilus natans (ATCC 13923)
Compatible ends
(5'…CTAG)
Ahl I, AspA2 I, AsuNH I, Avr II, Bcu I, Bln I, Nhe I, Xba I, XmaJ I
Isoschizomers
: Ahl I, Bcu I
Methylation Sensitivity
: Not sensitive to dam, dcm and CpG methylation
Unit definition :
One Unit is defined as the amount of Spe I required to completely digest 1 ㎍ of Ad-2 DNA in one hour at 37℃ in 50 ㎕ assay mixture.
Typical Reaction Conditions
: Add 5 ㎕ of 10X Reaction Buffer 'B'[final conc.: 6 mM Tris-HCl(pH 7.5 @ 37℃), 6 mM MgCl2, 50 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Spe 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 |
|
Activity(%) |
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 Spe 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
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φX174
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pBR322
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M13mp18
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SV40
|
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0
|
3
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0
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0
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0
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0
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0
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Spe I for one hour was showing after incubation of 1 ㎍ of Ad-2 DNA with units of Spe 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 Ad-2 DNA fragments were ligated with T4 DNA ligase at 16℃ and % of the ligated fragments could be recut with Spe I.
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Notes
- Conditions of low ionic strength, high enzyme concentration, glycerol > 5%, or pH > 8.0 may result in star activity.
- Spe I cleaves to leave a 5'…CTAG which can be ligated to fragments generated by Avr II, Nhe I, Sty I (C/CTAGG), and Xba I.
- Incubation of Spe I at 45°C results in an increase in activity. On the other hand, it is only 0~25% active at 25°C.
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