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Source
: Haemophilus influenzae Rd (exo- mutant)
(ATCC 51907)
Compatible ends
(5'…AGCT) & Isoschizomers
: Bbr I, BspKT8 I, BstF I, EcoV Ⅲ, Hsu I, LlaC I, Ssb I
Methylation Sensitivity
: Not sensitive to dam, dcm and CpG methylation
Unit definition :
One Unit is defined as the amount of Hind 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 'B'[final conc.: 6 mM Tris-HCl(pH 7.5 @ 37℃), 6 mM MgCl2, 50 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Hind Ⅲ 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(%) |
10~50 |
75~100 |
75~100 |
10~50 |
|
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 Hind 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, 100 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.
No. of Cleavage Sites
|
λ
|
Ad2
|
pUC18
|
φX174
|
pBR322
|
M13mp18
|
SV40
|
|
6
|
12
|
1
|
0
|
1
|
1
|
6
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Hind Ⅲ for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Hind 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 Hind Ⅲ.
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References
Hsu, M., and Berg, P. Biochemistry. Biochemistry 17: 131-138, (1978)/ Old, R., Murray, K., Roizes, G. J. Mol. Biol. 92: 331-339 (1975)
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