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Ecori enzyme

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EcoRI is a type II restriction enzyme that recognizes and cleaves the DNA sequence 5'-GAATTC-3'. It is commonly used in molecular biology and genetic engineering applications for the digestion and manipulation of DNA.

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Lab products found in correlation

Dna ladder, by Thermo Fisher Scientific (2 mentions) Midi kit, by Qiagen (2 mentions)

3 protocols using ecori enzyme

1

Plasmid Characterization of ESBL-Positive Transconjugants

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E. coli transconjugants resulting from conjugation between an ESBL-positive S. sonnei isolate and E. coli J53 (sodium azide resistance) were subjected to plasmid extraction using a plasmid Midi kit (Qiagen). For plasmid digestion, 500ng of each extracted plasmid DNA was digested with EcoRI enzyme (10 U/μl) (Fermentas), followed by electrophoresis on 0.8% agarose gel at 100V for 4 hours with 1 kb plus DNA ladder (Invitrogen). Plasmid restriction patterns were compared, and cluster analysis was performed using the UPGMA method and Jukes-Cantor correction using Bionumerics v5.1 software. For plasmid sequencing, 50ng of each plasmid DNA was subjected to library construction with a Nextera kit and sequenced using the MiSeq Illumina platform to generate 2x250 bp paired-end reads. De novo assembly was subsequently performed using SPADES v3.11 47 (link) and assembled contigs were annotated using Prokka v1.11 42 (link).
Duy P.T., Thi Nguyen T.N., Duong V.T., Chung The H., Alcock F., Boinett C., Ho Ngoc Dan T., Ha Thanh T., Thwaites G.E., Rabaa M.A, & Baker S. (2020). Commensal E. coli are a reservoir for the transfer of XDR plasmids into epidemic fluoroquinolone-resistant Shigella sonnei. Nature microbiology, 5(2), 256-264.
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2

Rat Cx46 Expression in Xenopus Oocytes

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Rat Cx46 pSP64T was kindly provided by Dr. Lisa Ebihara, Rosalind Franklin University. Cx46 DNA was linearized using the EcoRI enzyme (Fermentas) and transcribed using the in vitro transcription kit SP6 mMessagemMachine systems (Ambion) following manufacturer’s instructions. Stage V and VI oocytes were selected, defolliculated and injected with 5 ng of the RNA plus Cx38 antisense to null endogenous expression of Cx3835 (link). Oocytes were incubated at 18 °C in ND96 medium containing (in mM): 96 NaCl, 1 KCl, 1 MgCl2, 10 HEPES and 1.8 CaCl2. Experiments were done 24–48 hrs post injection.
Pinto B.I., Pupo A., García I.E., Mena-Ulecia K., Martínez A.D., Latorre R, & Gonzalez C. (2017). Calcium binding and voltage gating in Cx46 hemichannels. Scientific Reports, 7, 15851.
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3

Plasmid Characterization of ESBL-Positive Transconjugants

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E. coli transconjugants resulting from conjugation between an ESBL-positive S. sonnei isolate and E. coli J53 (sodium azide resistance) were subjected to plasmid extraction using a plasmid Midi kit (Qiagen). For plasmid digestion, 500ng of each extracted plasmid DNA was digested with EcoRI enzyme (10 U/μl) (Fermentas), followed by electrophoresis on 0.8% agarose gel at 100V for 4 hours with 1 kb plus DNA ladder (Invitrogen). Plasmid restriction patterns were compared, and cluster analysis was performed using the UPGMA method and Jukes-Cantor correction using Bionumerics v5.1 software. For plasmid sequencing, 50ng of each plasmid DNA was subjected to library construction with a Nextera kit and sequenced using the MiSeq Illumina platform to generate 2x250 bp paired-end reads. De novo assembly was subsequently performed using SPADES v3.11 47 (link) and assembled contigs were annotated using Prokka v1.11 42 (link).
Duy P.T., Thi Nguyen T.N., Duong V.T., Chung The H., Alcock F., Boinett C., Ho Ngoc Dan T., Ha Thanh T., Thwaites G.E., Rabaa M.A, & Baker S. (2020). Commensal E. coli are a reservoir for the transfer of XDR plasmids into epidemic fluoroquinolone-resistant Shigella sonnei. Nature microbiology, 5(2), 256-264.
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