Store at -20℃. For Research Use Only!

Nde  I

FauND I

Supplied with : 10X Reaction Buffer 'D'

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Neisseria denitrificans  (NRCC 31009)

Compatible ends (5'…TA)

Ase I, Bfa I, Csp6 I, CviQ I, FauND I, FspB I, Mae I, Mse I, PshB I, Tru1 I, Tru9 I, Vsp I, Xsp I

Isoschizomers : FauND I

Methylation Sensitivity

: Not sensitive to dam, dcm and CpG methylation

Unit definition : One Unit is defined as the amount of Nde 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 'D'[final conc.: 6 mM Tris-HCl(pH 7.9 @ 37℃), 6 mM MgCl₂, 150 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Nde 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
Activity(%)	<10	<10	10~50	75~100

Activity in a complete PCR Mix : 50~75%
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 Nde 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℃), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 200 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.

No. of Cleavage Sites

λ	Ad2	pUC18	φX174	pBR322	M13mp18	SV40
7	2	1	0	1	3	2

Quality control

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

Notes

- High glycerol concentration(more than 10%) may result in star activity.

- Ligation of DNA fragments digested Nde I is less efficient than other 2-base cohesive ends.

Therefore, more efficient ligation can be achieved by using the reaction conditions for blunt end ligation.

- Extended digestion is not recommended because the half-life of Nde I at 37℃ is only 15 minutes.

References

Watson, R.J., Schildkraut, I., Qiang, B.-Q., Martin, S.M., Visentin, L.P. FEBS Lett. 150:114-116 (1982)

Beams Biotechnology Co., Ltd.