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Source
: Acetobacter aceti IFO 3281
Compatible ends
(ACGT…3') : Tai I, Tsc I
Isoschizomers
: Ssp5230 I
- neoschizomers : Zra I
Methylation Sensitivity
: Not sensitive to dam and dcm methylation
: Blocked by CpG methylation
Unit definition :
One Unit is defined as the amount of Aat II 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 '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 Aat 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 |
F |
|
Activity(%) |
50~75 |
10~50 |
<10 |
<10 |
75~100 |
|
Activity in a complete PCR Mix : 10~50%
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 Aat 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), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 200 ㎍/㎖ 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
|
|
10
|
3
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1
|
1
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1
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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 Aat II for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Aat 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 λ DNA fragments were ligated with T4 DNA ligase at 16℃ and % of the ligated fragments could be recut with Aat II.
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References
Roberts, R.J., Nucleic Acid Res., 11: r135-r167 (1983)/ Sato, H., Suzuki, T., Yamada, Y., Agric. Biol. Chem., 54: 3319-3325 (1990)
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