Storage: −20°C
UNSPSC Code: 12352200
Components: Enzyme Solution; SuRE/Cut Buffer A 10x concentrated
RIDADR: NONH for all modes of transport
Analysis Note:
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.
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).
General description:
Sma I recognizes the sequence *C°C*C↓GGG and generates fragments with blunt ends.
Other Notes:
For life science research only. Not for use in diagnostic procedures.
Quality:
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.
DNA Profile:
Number of cleavage sites on different DNAs
• λ: 3
• φX174: 0
• Ad2: 12
• M13mp7: 0
• pBR322: 0
• pBR328: 0
• pUC18: 1
• SV40: 0
Specificity:
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).