Store at -20℃. For Research Use Only!

★   CpG

Age  I

AsiG I, BshT I, PinA I

Supplied with : 10X Reaction Buffer 'Age I'

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Agrobacterium  gelatinovorum  IAM 12617

Compatible ends (5'…CCGG)

Acc Ⅲ, Aco I, AsiG I, Bet I, Blf I, BsaW I, Bse118 I, BseA I, BshT I, Bsp13 I, BspE I,BsrF I, Cfr9 I, Cfr10 I, Kpn2 I, Mro I, MroN I, NgoM Ⅳ, PinA I, SgrA I, TspM I, Xma I, XmaC I

Isoschizomers : AsiG I, BshT I, CspA I, PinA I

Methylation Sensitivity

: Not sensitive to dam and dcm methylation

: Blocked by CpG methylation

Unit definition : One Unit is defined as the amount of Age 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 'Age I'[final conc.: 10 mM Bis-Tris-Propane-HCl (pH7.0 @ 25℃), 10 mM MgCl₂, 50 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Age I and 42~43 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 37℃, but more stable at 25℃.

Heat Inactivation : 65℃ for 20 min.

Activity in BeamsBio™ Buffer System

Rxn Buffer	A	B	C	D	'Age I'
Activity(%)	10~50	10~50	10~50	50-75	75~100

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 Age 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℃), 250 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.15% Triton™ X-100, 200 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.

No. of Cleavage Sites

λ	Ad2	pUC18	φX174	pBR322	M13mp18	pACYC177
13	5	0	0	0	0	2

Quality control

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

Notes

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

- Ends generated with Age I can be directly ligated to ends generated using Acc III, NgoM IV or Xma I.

- For extended digests use room temperature. Age I is 100% active and more stable in reaction at 25°C.

References

Yamada, Y., Mizuno, H., Sato, H., Akagawa, M., Yamasato, K., Agric. Biol. Chem., 53(6), 1747 (1989)/ Mizuno, H., Suzuki, T., Yamada, Y., Agric. Biol. Chem., 54(7), 1797 (1990)

Beams Biotechnology Co., Ltd.