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Source
: Streptomyces albus G (ATCC 49789)
Compatible ends
(5'…TCGA)
BssH I, PaeR7 I, PspX I, Sfr274 I, Sla I, Str I, Tli I, Xho I
Isoschizomers
: BspMK I
Methylation Sensitivity
: Not sensitive to dam and dcm methylation
: Blocked by CpG methylation
Unit definition :
One Unit is defined as the amount of Sal I required to completely digest 1 ㎍ of λ DNA (Hind III digest) in one hour at 37℃ in 50 ㎕ assay mixture.
Typical Reaction Conditions
: Add 5 ㎕ of 10X Reaction Buffer 'D'[final conc.: 6 mM Tris-HCl(pH 7.9 @ 37℃), 6 mM MgCl2, 150 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of Sal 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(%) |
<10 |
10~50 |
10~50 |
75~100 |
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Activity in a complete PCR Mix : less than 10%
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 Sal 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℃), 400 mM KCl, 0.1 mM EDTA, 1 mM DTT, 100 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.
No. of Cleavage Sites
|
λ
|
Ad2
|
pUC19
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φX174
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pBR322
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M13mp18
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SV40
|
|
2
|
3
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1
|
0
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1
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1
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0
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Quality control
Overdigestion: The same band pattern as a digestion in one unit of Sal I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of Sal 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 Sal I.
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Notes
- Conditions of low ionic strength, high enzyme concentration, glycerol > 5%, or pH > 8.0 may result in star activity.
- Supercoiled plasmids(e.g. pBR322 and pUC) may require up to 10-fold more Sal I for complete digestion than linear DNAs.
- When cleaving close to the end of DNA fragments, cleavage should be done at 37°C for 1 hour using 10 units/µg of DNA
with a minimum of 3 bases on each side of the recognition sequence.
- Sal I activity decreases if the reaction buffer pH is not between 7.8 and 8.0.
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References
Arrand, J.R., Myers, P.A., Roberts, R.J. J. Mol. Biol. 118: 113-122 (1978)/ Qiang, B.-Q., Schildkraut, I. Methods Enzymol. 155: 15-21 (1987)
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