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Source
: Streptomyces achromogenes ATCC 12767
Compatible ends
(GC…3') & Isoschizomers
: Cfr42 I, Ksp I, Sfr303 I, SgrB I, Sst II
Methylation Sensitivity
: Not sensitive to dam and dcm methylation
: Blocked by CpG methylation
Unit definition :
One Unit is defined as the amount of Sac II required to completely digest 1 ㎍ of Adenovirus-2 DNA in one hour at 37℃ in 50 ㎕ assay mixture.
Typical Reaction Conditions
: Add 5 ㎕ of 10X Reaction Buffer 'C'[final conc.: 10 mM Tris-HCl(pH 7.9 @ 37℃), 10 mM MgCl2, 50 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Sac II 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 |
50~75 |
75~100 |
50~75 |
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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 II. And then incubate at 37℃ for one hour. Relative activity is determined by gel electrophoresis.
Storage Conditions
: 10 mM Tris-HCl(pH 7.4 @ 25℃), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 500 ㎍/㎖ 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
|
|
4
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33
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0
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1
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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 Sac II for one hour was showing after incubation of 1 ㎍ of Adenovirus-2 DNA with units of Sac II 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 Adenovirus-2 DNA fragments were ligated with T4 DNA ligase at 16℃ and % of the ligated fragments could be recut with Sac II.
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Notes
- Sac II is able to cleave at 4 sites of λDNA, but the site positioned No. 40386 is difficult to be cleaved.
- Moderate levels of salt can inhibit Sac II. 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.
- Both incubations in BeamsBio™ Buffer 'A' and 'C' result 75~100% active, however, for use with supercoiled plasmid
in BeamsBio™ Buffer 'A' exhibits an incomplete digestion. So we recommend a BeamsBio™ Buffer 'C'.
- Buffer A is not recommended for use with supercoiled plasmid due to incomplete cutting.
- Sac II exhibits strong site preference. Sac II requires two recognition sites to activate cleavage.
Try reducing the reaction volume to 10 times the volume of enzyme added, or adding 30 units/㎍ of DNA and incubating
for 2~3 hours. Adding an oligonucleotide which contains the sequence will help if there is only a single site on the target DNA.
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