Store at -20℃. For Research Use Only!

CpG

Nar  I

Mly113 I

Supplied with : 10X Reaction Buffer 'Nar I'

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Nocardia  argentinensis  (ATCC 31306)

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, Hpa II, Hsp92 I, HpyCH4 Ⅳ, HspA I, Mae II, Mly113 I, Msp I, Nsp V, Psp1406 I, Ssi I, Taq I

Isoschizomers : Mly113 I

- neoschizomers : Bbe I, Din I, Ege I, Ehe I, Kas I, Sfo I

Methylation Sensitivity

: Not sensitive to dam and dcm methylation

: Blocked by CpG methylation

Unit definition : One Unit is defined as the amount of Nar I required to completely digest 1 ㎍ of φX174 RF I DNA in one hour at 37℃ in 50 ㎕ assay mixture.

Typical Reaction Conditions : Add 5 ㎕ of 10X Reaction Buffer 'Nar I'[final conc.: 50 mM Tris-HCl (pH 8.2 @ 25℃), 5 mM MgCl₂], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Nar I 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	'Nar I'
Activity(%)	75~100	50~75	75~100	10~50	75~100

Activity in a complete PCR Mix : almost 0%
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 Nar I. And then incubate at 37℃ for one hour. Relative activity is determined by gel electrophoresis.

Storage Conditions : 10 mM Tris-HCl (pH 7.4 @ 25℃), 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.15% Triton™ X-100, 500 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.

No. of Cleavage Sites

λ	Ad2	pUC18	φX174	pBR322	M13mp18	SV40
1	20	1	2	4	1	0

Quality control

Overdigestion: The same band pattern as a digestion in one unit of Nar I for one hour was showing after incubation of 1 ㎍ of φX174 RF I DNA with         units of Nar 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 φX174 RF I DNA fragments were ligated with T4 DNA ligase at 16℃ and        % of the ligated fragments could be recut with Nar I.

Notes

- Complete cleavage of pBR322 DNA by Nar I is very slow, because the site positioned No. 548 of the 4 positions on pBR322

DNA is difficult to be cleaved. The 5–10 fold amount of that amount of enzyme must be used, which is necessary for the

complete cleavage of pBR322 DNA.

References

Roberts, R.J. Nucleic Acids Res. 10: r117-r144 (1982)

Beams Biotechnology Co., Ltd.