Store at -20℃. For Research Use Only!

dcm   CpG   ★   50℃

Sfi  I

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

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Streptomyces fimbriatus (ATCC 15051)

Compatible ends (NNN…3')

AccB7 I, Ade I, Afi I, AlwN I, Bgl I, BsaX I, Bsc4 I, BseL I, BsiY I, Bsl I, BstAP I, BstMW I, Cai I, Dra III, Mwo I, PflB I, PflM I, Van91 I

Isoschizomers : Sdi I

Methylation Sensitivity

: Not sensitive to dam methylation

: Affected by overlapping dcm methylation

: Blocked by some combinations of overlapping CpG methylation

Unit definition : One Unit is defined as the amount of Sfi I required to completely digest 1 ㎍ of Ad-2 DNA in one hour at 50℃ 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 ㎍/㎕), 5 ㎕ of 10X BSA(final conc.: 100 ㎍/㎖), 1 ㎕ of Sfi I and 37~38 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 50℃.

Heat Inactivation : No

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℃.

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 : 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 Sfi I. And then incubate at 50℃ for one hour. Relative activity is determined by gel electrophoresis.

No. of Cleavage Sites

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

Quality control

Overdigestion: The same band pattern as a digestion in one unit of Sfi I for one hour was showing after incubation of 1 ㎍ of Ad-2 DNA with         units of Sfi I for 16 hr in 50 ㎕ of reaction mixture at 50℃ as determined by agarose gel electrophoresis.

Ligation-Recut: After a 10-fold digestion for one hour,        % of Ad-2 DNA fragments were ligated with T4 DNA ligase at 16℃ and        % of the ligated fragments could be recut with Sfi I.

Notes

- Sfi I is not inactivated by heating and therefore should be extracted with phenol/chloroform and once with chloroform,

and then precipitate the DNA with ethanol.

- Sfi I requires two copies of its recognition sequence for cleavage to occur. The two sites can be on either the same or

different DNA molecules.

- Sfi I shows star activity in the presence of Mn2+.

- Sfi I exhibits <10% activity at 37°C.

References

Qiang, B.-Q., Schildkraut, I. Nucleic Acids Res. 12: 4507-4516, (1984)/ Wentzell, L.M., et. al J. Mol. Biol. 248: 581-595 (1995)

Beams Biotechnology Co., Ltd.