Store at -20℃. For Research Use Only!

Fok  I

Supplied with : 10X Reaction Buffer 'B'

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Flavobacterium  okeanokoites  IFO 12536

Compatible ends (5'…NNNN)

Aar I, Acc36 I, Alw26 I, Bbs I, Bbv I, BfuA I, Bpi I, BpuA I, Bsa I, BseX I, BslF I, BsmA I, BsmB I, BsmF I, Bso31 I, BspM I, BspTN I, BstMA I, BstV1 I, BstV2 I, BtgZ I, Bve I, Eco31 I, Esp3 I, Faq I, Lwe I, SfaN I

Isoschizomers : Not known.

Methylation Sensitivity

: Not sensitive to dam, dcm and CpG methylation

Unit definition : One Unit is defined as the amount of Fok 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 'B'[final conc.: 6 mM Tris-HCl(pH 7.5 @ 37℃), 6 mM MgCl₂, 50 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Fok 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(%)	75~100	75~100	75~100	10~50

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 Fok 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℃), 50 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
150	78	5	8	12	4	11

Quality control

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

Notes

- Overdigestions of more than 5 units per ㎍ of DNA and incubation times more than 2 hours are not recommended,

because Fok I is not stable in reaction and it can degrade DNA.

References

Sugisaki, H., Kanazawa, S. Gene 16: 73-78 (1981)/ Kita, K., Kotani, H., Hiraoka, N., Nakamura, T., Yonaha, K. Nucleic Acids Res. 17: 8741-8753 (1989)

Beams Biotechnology Co., Ltd.