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Smai restriction endonuclease

Manufactured by New England Biolabs
Sourced in United States

SmaI is a type II restriction endonuclease that recognizes and cleaves the palindromic DNA sequence 5'-CCCGGG-3'. It generates blunt-ended DNA fragments.

Automatically generated - may contain errors

3 protocols using smai restriction endonuclease

1

Clonality Analysis of MRSA and Klebsiella

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The clonality of MRSA and meropenem-resistant isolates of Klebsiella pneumoniae detected at the Department of Anesthesiology and Intensive Care Medicine was assessed with pulsed-field gel electrophoresis (PFGE). Bacterial DNA extracted with a technique described by Husičková et al. [19 (link)] was digested by the XbaI restriction endonuclease (New England Biolabs, Ipswitch, MA, USA) for 24 h at 37 °C in Klebsiella pneumoniae isolates and by the SmaI restriction endonuclease (New England Biolabs, Ipswitch, MA, USA) for 24 h at 25 °C in Staphylococcus aureus strains. The obtained DNA fragments were separated by PFGE on 1.2% agarose gel for 24 h at 6 V/cm and pulse times of 2–35 s for both Klebsiella pneumoniae and Staphylococcus aureus strains. Subsequently, the gel was stained with ethidium bromide. The resulting restriction profiles were analyzed with the GelCompar II software (Applied Maths, Kortrijk, Belgium) using the Dice coefficient (1.2%) for comparing similarity and unweighted pair group method with arithmetic means for cluster analysis. The results were interpreted according to criteria described by Tenover et al. [20 (link)].
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2

Assessing S. aureus Biofilm Clonality

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To evaluate the clonality, a representative sample of trypsin‐inducible and trypsin‐uninducible biofilms isolates from each body part was prepared. Genotyping of 50 isolates was made by a PFGE protocol for S. aureus described by The Centre for Disease Control and Prevention, Atlanta, USA. Chromosomal DNA of each strain was extracted and digested with SmaI restriction endonuclease (New England Biolabs). Restriction fragments were resolved in a CHEF GenePath System (Bio‐Rad®). Classification of the clones was based on Tenover criteria (Tenover et al., 1995), and the percentage of relatedness was determined by the Dice coefficient (Dice, 1945). Isolates with cut‐off values of 85% were considered as belonging to the same clone.
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3

PFGE-Based Strain Similarity Analysis

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The similarity of the VRE isolates was performed with pulsed-field gel electrophoresis (PFGE). Bacterial DNA was isolated using a technique described by Husickova et al. and digested by the SmaI restriction endonuclease (New England Biolabs, Ipswich, MA, USA) for 24 h at 25 °C [33 (link)]. The obtained DNA fragments were separated by PFGE on 1.2% agarose gel for 24 h at 6 V/cm and pulse times of 2–35 s. Subsequently, the gel was stained with ethidium bromide. The resulting restriction profiles were analyzed with GelCompar II software (Applied Maths, Kortrijk, Belgium). The coefficient of similarity (CS) was calculated using the Dice algorithm. Individual clusters were analyzed with the unweighted pair group method with arithmetic mean algorithm, and the results were interpreted using the criteria defined by Tenover et al. [34 (link)]. Optimization and band matching tolerance was set at 1.2%.
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