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Analysis Note

Compatible endsSma I generates ends that are compatible with any blunt end.IsoschizomersThe enzyme is an isoschizomer to Cfr9 I, PspA I, Xma I, and XmaC I.Methylation sensitivitySma 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°CPFGE testedSma 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 assaySma 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 SystemThe 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.

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).

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

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 activities1µg 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 activityApproximately 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.

Specificity

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

Unit Definition

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.