Sma I

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

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).

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).

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.

Number of cleavage sites on different DNAs

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

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.

Do not store below −25°C

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.

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