Store at -20℃. For Research Use Only!

dam

Mbo  II

Supplied with : 10X Reaction Buffer 'B'

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Moraxella  bovis  (ATCC 10900)

Compatible ends (N…3')

Ahd I, AspE I, AsuHP I, BciV I, Bfi I, Bfu I, BmeR I, Bmu I, Bmr I, Bst4C I, Dri I, Eam1105 I, EclHK I, Hph I, Hpy188 I, HpyA V, HpyCH4 III, Mnl I, Taa I, Xcm I

Isoschizomers : Not known.

Methylation Sensitivity

: Blocked by overlapping dam methylation

: Not sensitive to dcm and CpG methylation

Unit definition : One Unit is defined as the amount of Mbo II required to completely digest 1 ㎍ of λ(dam^-) 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 Mbo 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
Activity(%)	10~50	75~100	50~75	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 Mbo 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 NaCl, 0.1 mM EDTA, 1 mM DTT, 500 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.

No. of Cleavage Sites

λ	Ad2	pUC18	φX174	pBR322	M13mp18	SV40
130	113	7	11	11	12	16

Quality control

Overdigestion: The same band pattern as a digestion in one unit of Mbo II for one hour was showing after incubation of 1 ㎍ of λ DNA with         units of Mbo 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 Mbo II.

Notes

- Single base extension(N…3') produced by Mbo II digestion is more difficult to ligate than blunt-end DNA fragments.

- Extended incubations longer than 1 hour are not recommended because Mbo II is not stable in reaction.

References

Gelinas, R.E., Myers, P.A., Roberts, R.J. J. Mol. Biol. 114: 169-179 (1977)/ Sektas, M., Kaczorowski, T., Podhajska, A.J. Nucleic Acids Res. 20: 433-438 (1992)

Beams Biotechnology Co., Ltd.