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Source
: Enterobacter aerogenes
Compatible ends
(5'…GGCC)
Aco I, BseX3 I, Bsp120 I, BstZ I, CciN I, Cfr I, Eag I, EclX I, Eco52 I, Gdi II, Not I, PspOM I, Xma III
Isoschizomers
: Aco I, Cfr I
Methylation Sensitivity
: Not sensitive to dam methylation
: Blocked by overlapping dcm and CpG methylation
Unit definition :
One Unit is defined as the amount of Eae 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 Eae 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 |
10~50 |
<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 Eae 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 KCl, 0.1 mM EDTA, 1 mM DTT, 200 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.
No. of Cleavage Sites
|
λ
|
Ad2
|
pUC18
|
φX174
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pBR322
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M13mp18
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SV40
|
|
39
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70
|
3
|
2
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6
|
3
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0
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Eae I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Eae 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 Eae I.
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