Store at -20℃. For Research Use Only!

★

Ase  I

PshB I, Vsp I

Supplied with : 10X Reaction Buffer 'Ase I'

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Aquaspirillum  serpens

Compatible ends (5'…TA)

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

Isoschizomers : PshB I, Vsp I

Methylation Sensitivity

: Not sensitive to dcm, dam and CpG methylation

Unit definition : One Unit is defined as the amount of Ase 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 'Ase I'[final conc.: 50 mM Tris-HCl (pH 7.9 @ 25℃), 10 mM MgCl₂, 100 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Ase 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	'Ase I'
Activity(%)	<10	75~100	75~100	50~75	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 Ase I. And then incubate at 37℃ for one hour. Relative activity is determined by gel electrophoresis.

Storage Conditions : 10 mM Tris-HCl (pH 7.5 @ 25℃), 500 mM KCl, 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
17	3	3	2	1	7	3

Quality control

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

Notes

- Conditions of low ionic strength, high enzyme concentration, glycerol concentration > 5%, or pH > 8.0 may result in star activity.

- Ase I cleaves pBR322 and Ad-2 DNA at a rate 3 times that of λ DNA.

References

Polisson, C., Morgan, R.D., Nucleic Acids Res., 16: 10365 (1988)/ Murakami, M., Yamada, Y., Agric. Biol. Chem., 54: 1791-1796 (1990)

Beams Biotechnology Co., Ltd.