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Source
: Acinetobacter calcoaceticus 65
Compatible ends
(5'…GTAC)
: Asp718 I, BsiW I, Pfl23 II, PspL I, Spl I, Sun I, Aau I, BsrG I, Bsp1407 I, BstAU I, SspB I, Tat I
Isoschizomers
: Asp718 I
- neoschizomers : Kpn I
Methylation Sensitivity
: Not sensitive to dam methylation
: Blocked by some combination of overlapping dcm methylation
: Blocked by some combination of overlapping CpG methylation
Unit definition :
One Unit is defined as the amount of Acc65 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 'D'[final conc.: 6 mM Tris-HCl (pH 7.9 @ 37℃), 6 mM MgCl2, 150 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 5 ㎕ of 10X BSA(final conc.: 100 ㎍/㎖), 1 ㎕ of Acc65 I and 37~38 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 37℃.
Heat Inactivation
: 65℃ for 20 min.
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Activity in BeamsBio™ Buffer System
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Rxn Buffer |
A |
B |
C |
D |
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Activity(%) |
10~50 |
50~75 |
75~100 |
75~100 |
Storage Conditions
: 10 mM Tris-HCl(pH 7.4), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 200 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.
No. of Cleavage Sites
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λ
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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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|
2
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8
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1
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0
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0
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1
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1
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Acc65 I for one hour was showing after incubation of 1 ㎍ of Ad-2 DNA with Units of Acc65 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 DNA fragments could be recut with Acc65 I.
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References
Prichodko, G.G., Rechnukova, N.I., Revin, V.E., Degtyarev, S.K., Sib. Biol. J. 1:59-60 (1991)
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