Store at -20℃. For Research Use Only!

Afl  II

Bfr I, BspT I, Bst98 I, BstAF I

Supplied with : 10X Reaction Buffer 'C', 10X BSA

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Anabaena flos-aquae CCAP 1403/13f

Compatible ends (5'…TTAA) & Isoschizomers

: Bfr I, BspT I, Bst98 I, BstAF I, MspC I, Vha464 I

Methylation Sensitivity

: Not sensitive to dcm, dam and CpG methylation

Unit definition : One Unit is defined as the amount of Afl II 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 'C'[final conc.: 10 mM Tris-HCl (pH 7.9 @ 37℃), 10 mM MgCl₂, 50 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 5 ㎕ of 10X BSA(final conc.: 100 ㎍/㎖), 1 ㎕ of Afl II and 37~38 ㎕ 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(%)	50~75	75~100	75~100	10~50

Activity in a complete PCR Mix : less than 10%
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 Afl II. And then incubate at 37℃ for one hour. Relative activity is determined by gel electrophoresis.

Storage Conditions : 10 mM Tris-HCl(pH 7.4), 50 mM KCl, 0.5 mM EDTA, 1 mM DTT, 200 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.

No. of Cleavage Sites

λ	Ad2	pUC18	φX174	pBR322	M13mp18	SV40
3	4	0	2	0	0	1

Quality control

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

Notes

- Ligation of DNA fragments digested Afl II is less efficient than other 4-base cohesive ends.

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

- Afl II is inhibited by salt concentrations greater than 50 mM.

References

Roberts, R.J., Nucleic Acid Res., 10: r117-r144 (1982)/ Whitehead, P. R., Brown, N. L., J. Gen. Micorbiol.. 131:951-958 (1985)

Beams Biotechnology Co., Ltd.