Store at -20℃. For Research Use Only!

dam   CpG

Mbo  I

Dpn II, Nde II, Kzo9 I, Sau3A I

Supplied with : 10X Reaction Buffer 'C'

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Moraxella  bovis  (ATCC 10900)

Compatible ends (5'…GATC)

BamH I, BfuC I, Bcl I, Bgl II, Bsp143 I, BstMB I, BstX2 I, BstY I, Dpn II, Fba I, Ksp22 I, Kzo9 I, Mfl I, Nde II, Psu I, Sau3A I, Xho II

Isoschizomers

: BfuCI, Bsp143 I, BssMI, BstMBI, DpnII, Kzo9 I, NdeII, Sau3A I

- neoschizomer : BstKT I

Methylation Sensitivity

: Blocked by dam methylation

: Not sensitive to dcm methylation

: Affected by overlapping CpG methylation

Unit definition : One Unit is defined as the amount of Mbo I 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 '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 Mbo I and 42~43 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 37℃.

Heat Inactivation : 65℃ for 20 min.

Storage Conditions : 10 mM Tris-HCl(pH 7.5 @ 25℃), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.15% Triton™ X-100, 100 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.

Activity in BeamsBio™ Buffer System

Rxn Buffer	A	B	C	D
Activity(%)	10~50	75~100	75~100	50~75

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 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
116	87	15	0	22	7	8

Quality control

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

Notes

- Isoschizomers of Mbo I are Nde II and Sau3A I. Mbo I and Nde II are sensitive to dam methylation.

On the other hands, Sau3A I is not sensitive to dam methylation.

References

Gelinas, R.E., Myers, P.A., Roberts, R.J. J. Mol. Biol. 114: 169-179 (1977)/ Dreiseikelmann, B., Eichenlaub, R., Wackernagel, W. Biochim. Biophys. Acta 562: 418-428 (1979)

Beams Biotechnology Co., Ltd.