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Source
: Acetobacter pasteurianus IFO 13753
Compatible ends
(5'…TGCA) & Isoschizomers
: Alw44 I, Vne I
Methylation Sensitivity
: Not sensitive to dam and dcm methylation
: Blocked by overlapping CpG methylation
Unit definition :
One Unit is defined as the amount of ApaL 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 'E'[final conc.: 20 mM Tris-acetate (pH 7.9 @ 25℃), 50 mM Potassium acetate, 10 mM Magnesium acetate, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 5 ㎕ of 10X BSA(final conc.: 100 ㎍/㎖), 1 ㎕ of ApaL I and 37~38 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 37℃.
Heat Inactivation
: No
Activity in BeamsBio™ Buffer System
|
Rxn Buffer |
A |
B |
C |
D |
E |
|
Activity(%) |
75~100 |
75~100 |
10~50 |
<10 |
75~100 |
|
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 ApaL 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), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 200 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.
No. of Cleavage Sites
|
λ
|
Ad2
|
pUC18
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φX174
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pBR322
|
M13mp18
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SV40
|
|
4
|
7
|
3
|
1
|
3
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0
|
0
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of ApaL I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of ApaL 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 ApaL I.
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References
Yamada, Y., Murakami, M., Agric. Biol. Chem., 49: 3627-3629 (1985)/ Murakami, M., Yamada, Y., Agric. Biol. Chem., 54: 1791-1796 (1990)
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