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Source
: Providencia stuartii 164 (ATCC 49762)
Compatible ends
(TGCA…3')
BspMA I, EcoT22 I, Mph1103 I, Nsi I, Sbf I, Sda I, Sse8387 I, Zsp2 I
Isoschizomers
: BspMA I
Methylation Sensitivity
: Not sensitive to dam, dcm and CpG methylation
Unit definition :
One Unit is defined as the amount of Pst 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 'Pst I'[final conc.: 90 mM Tris-HCl(pH 7.5 @ 37℃), 10 mM MgCl2, 50 mM NaCl], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Pst 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 |
'Pst I' |
|
Activity(%) |
10~50 |
50~75 |
50~75 |
50~75 |
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 Pst 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 NaCl, 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
|
pUC19
|
φX174
|
pBR322
|
M13mp18
|
SV40
|
|
28
|
30
|
1
|
1
|
1
|
1
|
2
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Pst I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Pst 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 Pst I.
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Notes
- Conditions of high pH, low salt, high glycerol(>12%), 8% DMSO can cause star activity.
- 0.5~2 mM spermidine inhibits digestion by Pst I. Spermidine suppresses star activity of Pst I.
- The presence of adjacent runs of G-C base pairs confers significant resistance to cleavage.
- 100% dUTP incoporation reduces Pst I cleavage to 25%.
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References
Smith, D.I. et al Nucleic Acids Res. 3: 343-353 (1976)/ Malyguine, E. et al Gene 8: 163-177 (1980)/ Armstrong, K., Bauer, W.R. Nucleic Acids Res. 10: 993-1007 (1982)/ Pingoud, A. Eur. J. Biochem. 147: 105-109 (1985)/ Kuosmanen, M., Poso, H. FEBS Lett. 179: 17-20 (1985)/ Glenn, T.C. et al Biotechniques 17: 1086-1090 (1994)
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