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Source : Bacillus amyloliquefaciens H ATCC 49763
Compatible ends (5'…GATC)
Bcl I, BfuC I, Bgl II, Bsp143 I, BstMB I, BstX2 I, BstY I, Dpn II, Fba I, Ksp22 I, Kzo9 I, Mbo I, Mfl I, Nde II, Psu I, Sau3A I, Xho II
Isoschizomers : Bst I
Methylation Sensitivity
: Not sensitive to dcm, dam and CpG methylation
Unit definition : One Unit is defined as the amount of BamH I 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 'BamH I'[final conc.: 10 mM Tris-HCl (pH 7.9 @ 25℃), 5 mM MgCl2, 100 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 5 ㎕ of 10X BSA(final conc.: 100 ㎍/㎖), 1 ㎕ of BamH I and 37~38 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 37℃.
Heat Inactivation : No
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℃.
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Activity in BeamsBio™ Buffer System
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Rxn Buffer |
A★ |
B |
C |
D |
'BamH I' |
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Activity(%) |
75~100 |
75~100 |
75~100 |
<10 |
75~100 |
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 BamH I. And then incubate at 37℃ for one hour. Relative activity is determined by gel electrophoresis.
No. of Cleavage Sites
|
λ |
Ad2 |
pUC18 |
φX174 |
pBR322 |
M13mp18 |
SV40 |
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5 |
3 |
1 |
0 |
1 |
1 |
1 |
Quality control
Overdigestion: The same band pattern as a digestion in one unit of BamH I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of BamH 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 λ DNA fragments were ligated with T4 DNA ligase at 16℃ and % of the ligated fragments could be recut with BamH I. |
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Notes
- Conditions of low ionic strength, high enzyme concetration, or glycerol conc. > 10% may result in star activity.
- Concentrations of BamH I as high as 10 U/㎍ DNA can be completely heat-inactivated by incubating the mixture for 15 min at
65℃. However, higher enzyme concentrations will not be completely inactivated under these conditions. Therefore should be
extracted with phenol/chloroform and once with chloroform, and then precipitate the DNA with ethanol. |
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References
Wilson, G.A., Young, F.E., J. Mol. Biol., 97: 123-125 (1975)/ Roberts, R.J., Wilson, G.A., Young, F.E., Nature, 265: 82-84 (1977)/ Wilson, G.A., Young, F.E., Methods Enzymol., 65: 147-153 (1980)
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