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Source
: Klebsiella pneumoniae OK8 (ATCC 49790)
Compatible ends
(GTAC…3')
: Kpn I has no compatible ends to other known restriction enzymes.
Isoschizomers
- neoschizomers : Acc65 I, Asp718 I
Methylation Sensitivity
: Not sensitive to dcm, dam and CpG methylation
Unit definition :
One Unit is defined as the amount of Kpn I required to completely digest 1 ㎍ of Ad-2 DNA in one hour at 37℃ in 50 ㎕ assay mixture.
Typical Reaction Conditions
: Add 5 ㎕ of 10X Reaction Buffer 'F'[final conc.: 10 mM Tris-HCl (pH7.5 @ 37℃), 7 mM MgCl2, 50 mM KCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Kpn I and 42~43 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 37℃.
Heat Inactivation
: No
Activity in BeamsBio™ Buffer System
|
Rxn Buffer |
A★ |
B |
C |
D |
F |
|
Activity(%) |
75~100 |
10~50 |
10~50 |
<10 |
75~100 |
|
Activity in a complete PCR Mix : 50~75%
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 Kpn 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 ℃), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.15% Triton™ X-100, 100 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.
No. of Cleavage Sites
|
λ
|
Ad2
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pUC18
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φX174
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pBR322
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M13mp18
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SV40
|
|
2
|
8
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1
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0
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0
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1
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1
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Kpn I for one hour was showing after incubation of 1 ㎍ of Ad-2 DNA with units of Kpn 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 Ad-2 DNA fragments were ligated with T4 DNA ligase at 16℃ and % of the ligated fragments could be recut with Kpn I.
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Notes
- Kpn 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.
- Kpn I shows star activity in reactions containing high glycerol(>5%, v/v), excess enzyme and high pH (>8.0).
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References
Tomassini, J., Roychoudhury, R., Wu, R., Roberts, R.J. Nucleic Acid Res 5: 4055-4064 (1978)/ Mural, R.J. Nucleic Acid Res. 15: 9085 (1987)
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