Store at -20℃. For Research Use Only!

★

Sca  I

Supplied with : 10X Reaction Buffer 'Sca I'

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Streptomyces  caespitosus

Compatible ends : with any blunt end

Isoschizomers : Ass I, BmcA I, Zrm I

Methylation Sensitivity

: Not sensitive to dcm, dam and CpG methylation

Unit definition : One Unit is defined as the amount of Sca 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 'Sca 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 Sca I and 42~43 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 37℃.

Heat Inactivation : 80℃ for 20 min.

Activity in BeamsBio™ Buffer System

Rxn Buffer	A	B★	C★	D	'Sca I'
Activity(%)	<10	75~100	50~75	75~100	75~100

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 Sca 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℃), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 500 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.

No. of Cleavage Sites

λ	Ad2	pUC18	φX174	pBR322	M13mp18	SV40
5	5	1	0	1	0	0

Quality control

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

Notes

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

- Incubations in BeamsBio™ Buffer 'B' and 'C' result 50~100% active, however, in these conditions Sca I exhibits

star activity. So we recommend an unique buffer 'Sca I'[final conc.: 50 mM Tris-HCl (pH 7.9 @ 25℃), 10 mM MgCl₂,

100 mM NaCl, 1 mM DTT].

- Supercoiled plasmids(e.g. pBR322 and pUC) may require up to 20 units of Sca I to cleave completely 1 µg of DNA.

References

Takahashi, H., Kojima, H., Saito, H. Biochem. J. 231: 229-232 (1985)

Beams Biotechnology Co., Ltd.