When 14 MDR-AB isolates were detected simultaneously during Jan-Mar, 2013, the genetic relatedness among those MDR-AB strains collected from the patients and the HTCS during the same period were further analyzed through pulsed field gel electrophoresis (PFGE) according to the protocol [29 (link)]. Briefly. Fresh and pure bacterial cultures were embedded in agarose plugs and digested with proteinase K (20 mg/mL), followed by ApaI restriction endonuclease (TaKaRa, Dalian, Beijing, China). The standard strain Salmonella enterica serotype Braenderup H9812 digested with XbaI was used as a marker. The electrophoresis was performed in 0.5 × TBE buffer in a pulsed-field electrophoresis system (Chef Mapper; Bio-Rad Laboratories, Hercules, CA, USA), and the conditions were as follows: 14 °C, 6 V/cm, switch angle 120°, switch ramp 5–20 s for 19 h. BioNumerics software version 7.6 (Applied Maths, Sint-Martens-Latem, Belgium) was used to analyze the PFGE banding patterns. A cut off of 85% was used to judge the relatedness of strains analyzed based on the tree constructed by the unweighted pair group method of averages and a position tolerance of 1.5%.
Apai restriction endonuclease
Manufactured by Takara Bio
Sourced in China
ApaI is a type II restriction endonuclease that recognizes and cleaves the palindromic DNA sequence 5'-GGGCCC-3'. It generates 5' overhangs upon cleavage.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using apai restriction endonuclease
1
Genetic Relatedness of MDR-AB Strains
2
PFGE Analysis of MDR-AB Strains
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When 14 MDR-AB isolates were detected simultaneously during Jan-Mar, 2013, the genetic relatedness among those MDR-AB strains collected from the patients and the HTCS during the same period were further analyzed through pulsed eld gel electrophoresis (PFGE) according to the protocol (29) . Brie y. Fresh and pure bacterial cultures were embedded in agarose plugs and digested with proteinase K (20 mg/mL), followed by ApaI restriction endonuclease (TaKaRa, Dalian, Beijing, China). The standard strain Salmonella enterica serotype Braenderup H9812 digested with XbaI was used as a marker. The electrophoresis was performed in 0.5 × TBE buffer in a pulsed-eld electrophoresis system (Chef Mapper; Bio-Rad Laboratories, Hercules, CA, USA), and the conditions were as follows: 14°C, 6 V/cm, switch angle 120°, switch ramp 5-20 s for 19 h. BioNumerics software version 7.6 (Applied Maths, Sint-Martens-Latem, Belgium) was used to analyze the PFGE banding patterns. A cut off of 85% was used to judge the relatedness of strains analyzed based on the tree constructed by the unweighted pair group method of averages and a position tolerance of 1.5%.
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