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Source : Haemophilus aegyptius ATCC 11116
Compatible ends : with any blunt end
Isoschizomers
: BshF I, Bsn I, BspAN I, BsuR I, Pho I
Methylation Sensitivity
: Not sensitive to dam, dcm and CpG methylation
Unit definition : One Unit is defined as the amount of Hae Ⅲ 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 '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 Hae Ⅲ and 42~43 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 37℃.
Heat Inactivation : 80℃ for 20 min.
Activity in BeamsBio™ Buffer System
|
Rxn Buffer |
A |
B |
C |
D |
|
Activity(%) |
75~100 |
75~100 |
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 Hae Ⅲ. 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℃), 400 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 |
pUC18 |
φX174 |
pBR322 |
M13mp18 |
SV40 |
|
149 |
216 |
11 |
11 |
22 |
15 |
19 |
Quality control
Overdigestion: The same band pattern as a digestion in one unit of Hae Ⅲ for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Hae Ⅲ 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 Hae Ⅲ. |
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References
Middleton, J.H., Edgell, M.H., Hutchison, C.A. Ⅲ J. Virol. 10: 42-50 (1972)/ Blakesley, R.W., Dodgson, J.B., Nes, I.F., Wells, R.D. J. Biol. Chem. 252: 7300-7306 (1977)
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