Store at -20℃. For Research Use Only!

CpG

Hpa  II

BsiS I, Hap II, Msp I

Supplied with : 10X Reaction Buffer 'A'

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Haemophilus parainfluenzae (ATCC 49669)

Compatible ends (5'…CG)

Aci I, Acl I, Acy I, BmeT110 I, Bpu14 I, Bsa29 I, BsaH I, Bsp119 I, BspAC I, BspD I, BspT104 I, BspX I, BstAC I, BstB I, Bsu15 I, Cla I, Csp45 I, Hap II, Hin1 I, Hin6 I, HinP1 I, Hsp92 I, HpyCH4 Ⅳ, HspA I, Mae II, Mly113 I, Msp I, Nar I, Nsp V, Psp1406 I, Ssi I, Taq I

Isoschizomers : BsiS I, Hap II, Msp I

Methylation Sensitivity

: Not sensitive to dam and dcm methylation

: Blocked by CpG methylation

Unit definition : One Unit is defined as the amount of Hpa II 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 'A'[final conc.: 6 mM Tris-HCl (pH 7.5 @ 37℃), 6 mM MgCl₂, 6 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Hpa II 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 7.4 @ 25℃), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 500 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.

Activity in BeamsBio™ Buffer System

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

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 Hpa II. 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
328	171	13	5	26	18	1

Quality control

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

Notes

- Hpa II does not cleave ^m5CCGG and C^m5CGG, whereas Msp I(an isoschizomer of Hpa II) is sensitive only to methylation

at the 5'-C-residue, and cleaves C^m5CGG but not ^m5CCGG.

- Hpa II is inhibited by salt concentrations above 50 mM KCl.

- The number of cleavage site for Hpa II in SV40 DNA is only one. The activity for this site is slow due to the enzyme

needing two sites to cut sufficiently.

References

Sharp, P.A., et al. J. Biochemistry 12: 3055-3063 (1973)/ Oller, R.A., Broek, V.W., Conrad, M., Topal, M.D. Biochemistry 30:2543-2549 (1991)

Beams Biotechnology Co., Ltd.