Store at -20℃. For Research Use Only!

dcm   CpG   30℃

Apa  I

Supplied with : 10X Reaction Buffer 'A'

Concentration :            units/㎕

Lot # :                

Expiration date :                

Source : Acetobacter pasteurianus NCIB 7215

Compatible ends (GGCC…3') : Ppe I

Isoschizomers

: Bsp1201 I(neoschizomer), PspOM I(neoschizomer)

Methylation Sensitivity

: Not sensitive to dam methylation

: Blocked by overlapping dcm methylation

: Blocked by overlapping CpG methylation

Unit definition : One Unit is defined as the amount of Apa I required to completely digest 1 ㎍ of pXba DNA in one hour at 30℃ in 50 ㎕ assay mixture.

Typical Reaction Conditions : Add 5 ㎕ of 10X Reaction Buffer 'A'[final conc.: 6 mM Tris-HCl (pH 7.5 @ 37℃), 6 mM MgCl₂, 6 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Apa I and 42~43 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 30℃.

Heat Inactivation : 65℃ for 20 min.

Activity in BeamsBio™ Buffer System

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

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 Apa I. And then incubate at 30℃ for one hour. Relative activity is determined by gel electrophoresis.

Storage Conditions : : 10 mM Tris-HCl(pH 7.4 @ 25℃), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.15% Triton™ X-100, 100 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.

No. of Cleavage Sites

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

Quality control

Overdigestion: The same band pattern as a digestion in one unit of Apa I for one hour was showing after incubation of 1 ㎍ of DNA with         units of Apa I for 16 hr in 50 ㎕ of reaction mixture at 30℃ as determined by agarose gel electrophoresis.

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

Notes

- Optimal activity is at 30~32°C. Incubation at 37°C results in 100% activity, however, the half-life of Apa I at 37°C is

only 30 minutes.

- Apa I is inhibited by salt concentrations above 50 mM.

References

Seurinck, J., Van de Voorde, A., Van Montagu, M., Nucleic Acids Res., 11: 4409-4415 (1983)/ Yamada, Y., Murakami, M., Agric. Biol. Chem., 49: 3627-3629 (1985)/ Larimer, F.W., Nucleic Acids Res., 15: 9087 (1987)

Beams Biotechnology Co., Ltd.