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Source
: Serratia marcescens (ATCC 49779)
Compatible ends
: with any blunt end
Isoschizomers
: CfrJ4 I, PaeB I, PspAL I
- neoschizomers : Cfr9 I, TspM I, Xma I, XmaC I
Methylation Sensitivity
: Not sensitive to dam and dcm methylation
: Blocked by CpG methylation
Unit definition :
One Unit is defined as the amount of Sma I required to completely digest 1 ㎍ of λ DNA in one hour at 25℃ in 50 ㎕ assay mixture.
Typical Reaction Conditions
: Add 5 ㎕ of 10X Reaction Buffer 'F'[final conc.: 10 mM Tris-HCl (pH 7.5 @ 37℃), 7 mM MgCl2, 50 mM KCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Sma I and 42~43 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 25℃.
Heat Inactivation
: 65℃ for 20 min.
Activity in BeamsBio™ Buffer System
|
Rxn Buffer |
A |
B |
C |
D |
F |
|
Activity(%) |
<10 |
<10 |
<10 |
<10 |
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 Sma I. And then incubate at 25℃ for one hour. Relative activity is determined by gel electrophoresis.
Storage Conditions
: 10 mM Tris-HCl(pH 7.5 @ 25℃), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 100 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.
No. of Cleavage Sites
|
λ
|
Ad2
|
pUC18
|
φX174
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pBR322
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M13mp18
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SV40
|
|
3
|
12
|
1
|
0
|
0
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1
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0
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Sma I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Sma I for 16 hr in 50 ㎕ of reaction mixture at 25℃ 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 Sma I.
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Notes
- Conditions of high enzyme concentration , glycerol concentration > 5% or pH > 8.0 may result in star activity.
- The reaction temperature for Sma I is 25°C. Incubation at 37°C gives 50% activity. Sma I has a half-life of 15 minutes at 37°C.
- Activities of Sma I in Reaction Buffer 'A', 'B', 'C' and 'D' are less than 10%, because Sma I requires potassium ion to work.
So we recommend a buffer 'F'[final conc.: 10 mM Tris-HCl (pH 7.5 @ 37℃), 7 mM MgCl2, 50 mM KCl, 1 mM DTT].
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References
Endow, S.A., Roberts, R.J. J. Mol. Biol. 112: 521-529 (1977)/ Youssoufian, H., Mulder, C. J. Mol. Biol. 150: 133-136 (1981)
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