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Source : Streptomyces achromogenes ATCC 12767
Compatible ends (AGCT…3') : Psp124B I, Sst I
Isoschizomers
: Psp124B I, Sst I
- neoschizomer : Ecl136 II, EcolCR I
Methylation Sensitivity
: Not sensitive to dam, dcm and CpG methylation
Unit definition : One Unit is defined as the amount of Sac 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 'A'[final conc.: 6 mM Tris-HCl (pH 7.5 @ 37℃), 6 mM MgCl2, 6 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Sac 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 |
10~50 |
10~50 |
<10 |
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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 Sac 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 |
pUC19 |
φX174 |
pBR322 |
M13mp18 |
SV40 |
|
2 |
16 |
1 |
0 |
0 |
1 |
0 |
Quality control
Overdigestion: The same band pattern as a digestion in one unit of Sac I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Sac 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 Sac I. |
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Notes
- Sac I is inhibited by salt concentrations > 10 mM. Mini-prep DNA containing residual salt is resistant to cleavage.
The DNA may need to be ethanol precipitated and washed with 70% ethanol to reduce the salt.
Drop dialysis is the most effective way of reducing the salt.
- Sac I is sensitive to cytosine methylation at GAGmCTC but not GAGCTmC and insensitive to adenine methylation at GmAGCTC.
- Supercoiled plasmids may require up to 5-fold more Sac I(5~10 U/㎍ of pDNA) for complete digestion than linear DNA.
- High pH(> 8.0) will inhibit Sac I. |
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References
Kang, S.C., Yoon, H., Yoo, O.J. Korean Biochem. J. 19: 41-46 (1986)/ Zhuravleva, L.I., Oreshkin, E.N., Bezborodov, A.M. Prikl. Biokhim. Mikrobiol. 23: 208-215 (1987)
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