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Source
: Rhodopseudomonas sphaeroides
Compatible ends
: with any blunt end
Isoschizomers
: Afa I
- neoschizomers : Csp6 I, CviQ I
Methylation Sensitivity
: Not sensitive to dam and dcm methylation
: Blocked by some combinations of overlapping CpG methylation
Unit definition :
One Unit is defined as the amount of Rsa 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 'C'[final conc.: 10 mM Tris-HCl(pH 7.9 @ 37℃), 10 mM MgCl2, 50 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Rsa 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 |
75~100 |
<10 |
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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 Rsa 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
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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
|
|
113
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83
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3
|
11
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3
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19
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12
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Rsa I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Rsa 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 Rsa I.
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References
Lynn, S.P., Cohen, L.K., Kaplan, S., Gardner, J.F. J. Bacteriol. 142, 380-383, (1980)
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