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Source
: Diplococcus pneumoniae
Compatible ends
: with any blunt end
Isoschizomers
: Cfu I, Mal I
- neoschizomers : BfuC I, Bsp143 I, BssM I, BstKT I, BstMB I,
Dpn II, Kzo9 I, Mbo I, Nde I, Sau3A I
Methylation Sensitivity
: Not sensitive to dcm and CpG methylation
: Dpn I does not cleave dam- DNA.
Unit definition :
One Unit is defined as the amount of Dpn I required to completely digest 1 ㎍ of pBR322(dam+) 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 Dpn 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 |
|
Activity(%) |
50~75 |
75~100 |
75~100 |
50-75 |
|
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 Dpn 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℃), 400 mM NaCl, 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
|
M13mp18
|
SV40
|
|
116
|
87
|
15
|
0
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22
|
7
|
8
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Dpn I for one hour was showing after incubation of 1 ㎍ of pBR322 DNA with units of Dpn 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 pBR322 DNA fragments were ligated with T4 DNA ligase at 16℃ and % of the ligated fragments could be recut with Dpn I.
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