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Source
: Haemophilus parainfluenzae (ATCC 49669)
Compatible ends
: with any blunt end
Isoschizomers
: KspA 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 Hpa 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 'F'[final conc.: 10 mM Tris-HCl (pH 7.5 @ 37℃), 7 mM MgCl2, 50 mM KCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Hpa 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(%) |
10~50 |
50~75 |
10~50 |
10~50 |
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 Hpa 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
|
pUC18
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φX174
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pBR322
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M13mp18
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SV40
|
|
14
|
6
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0
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3
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0
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0
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4
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Hpa I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Hpa 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 Hpa I.
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Notes
- Conditions of high enzyme concentration , glycerol concentration > 5% or pH > 8.0 may result in star activity.
- Spermidine in concentrations of 0.5~2.0 mM inhibit Hpa I activity.
- Hpa 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.
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References
Sharp, P.A., Sugden, B., Sambrook, J. Biochemistry 12: 3055-3063 (1973)/ Kuosmanen, M., Poso, H. FEBS Lett. 179: 17-20 (1985)
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