SMAI-RO

Sma I

from Serratia marcescens Sb

• UNSPSC Code: 12352204
• Specific activity: 10000 U/mL
• Biological source: Serratia marcescens
• Concentration: <0.1 % (w/w)

Propiedades

• biological source: Serratia marcescens
• form: solution
• specific activity: 10000 U/mL
• packaging: pkg of 1,000 U (10220566001 [10 U/μl]), pkg of 5,000 U (10656348001 [10 U/μl]), pkg of 5,000 U (11047639001 [40 U/μl])
• manufacturer/tradename: Roche
• concentration: <0.1 % (w/w)
• parameter: 25 °C optimum reaction temp.
• technique(s): electrophoresis: suitable
• color: colorless
• pH: 7.0 (39 °F)
• solubility: water: miscible
• suitability: suitable for molecular biology
• application(s): life science and biopharma
• foreign activity: Endonucleases, none detected (up to 20 U with lambda-DNA), Endonucleases, none detected (up to 20U with pBR 322-DNA )
• shipped in: dry ice
• storage temp.: −20°C

Descripción

General description

Sma I recognizes the sequence *C°C*C↓GGG and generates fragments with blunt ends.

Application

Sma I has been used in macrorestriction analysis. It has also been used in the restriction enzyme mixture during restriction digestion, amid rapid pulsed-field gel electrophoresis (PFGE).

Biochem/physiol Actions

Recognition sites: *C °C*CGGG
*C °C*CGGG
Restriction site: *C °C*C↓GGG
*C °C*C↓GGG
Heat inactivation: Sma I can be heat inactivated by incubation at 65 °C for 15 minutes (up to 100 U/μg DNA).

Number of cleavage sites on different DNAs

• λ: 3
• φX174: 0
• Ad2: 12
• M13mp7: 0
• pBR322: 0
• pBR328: 0
• pUC18: 1
• SV40: 0

Preparation Note

Do not store below −25°C

Analysis Note

Absence of nonspecific endonuclease activities
1μg λDNA is incubated for 16 hours in 50μl SuRE/Cut Buffer A with an excess of Sma I. The number of enzyme units which do not change the enzyme-specific pattern is stated in the certificate of analysis.

Absence of exonuclease activity
Approximately 5μg [3H] labeled calf thymus DNA are incubated with 3μl Sma I for 4 hours at +37°C in a total volume of 100μl 50mM Tris-HCl, 10mM MgCl₂, 1mM Dithioerythritol, pH approximately 7.5. Under these conditions, no release of radioactivity is detectable, as stated in the certificate of analysis.

Compatible ends
Sma I generates ends that are compatible with any blunt end.

Isoschizomers
The enzyme is an isoschizomer to Cfr9 I, PspA I, Xma I, and XmaC I.

Methylation sensitivity
Sma I is not inhibited by 5-methylcytosine at the middle of the three C residues (°) in the recognition sequence. However, the activity is inhibited by 5-methylcytosine at the other Cs (*) or 4-methylcytosine in any position within the recognition sequence (*C°C*C↓GGG).

Incubation temperature
+25°C

PFGE tested
Sma I has been tested in Pulsed-Field Gel Electrophoresis (on bacterial chromosomes). For cleavage of genomic DNA (E. coli C 600) embedded in agarose for PFGE analysis, we recommend 10 U of enzyme/μg DNA and 4 hour incubation time.

Ligation and recutting assay
Sma I fragments obtained by complete digestion of 1μg λDNA are ligated with 1U T4 DNA Ligase in a volume of 10μl by incubation for 16 hours at +25°C in 66mM Tris-HCl, 5mM MgCl₂, 5mM Dithioerythritol, and 1mM ATP, pH 7.5 (at +20°C) resulting in >80% recovery of 1μg λDNA fragments.
Subsequent re-cutting with Sma I yields >80% of the typical pattern of λDNA × Sma I fragments.

SuRE/Cut Buffer System
The buffer in bold is recommended for optimal activity

• A: 100%
• B: 0-10%
• H: 0-10%
• L: 0-10%
• M: 0-10%

Activity in PCR buffer: 100%

Relative activity in PCR mix (Taq DNA Polymerase buffer) is 100%. The PCR mix contained λDNA, primers, 10 mM Tris-HCl (pH 8.3, 20 °C), 50 mM KCl, 1.5 mM MgCl₂, 200 μM dNTPs, 2.5 U Taq DNA polymerase. The mix was subjected to 25 amplification cycles.Activity in reaction buffer of Pwo SuperYield DNA Polymerase PCR Mix is 100%. If supplemented with GC-RICH Solution, activity remains at 100%. Pwo SuperYield DNA Polymerase PCR Mix is not available in U.S.

Other Notes

For life science research only. Not for use in diagnostic procedures.

One unit is the enzyme activity that completely cleaves 1 μg λDNA in one hour at +25 °C in a total volume of 25 μl (1x) SuRE/Cut buffer A.

Solo componentes del kit

• Enzyme Solution
• SuRE/Cut Buffer A 10x concentrated

Información de seguridad

• Clase de almacenamiento: 12 - Non Combustible Liquids
• wgk: WGK 1
• flash_point_f: does not flash
• flash_point_c: does not flash