Plasmid from the library was digested using SalI enzyme, and the digestion products were separated by gel electrophoresis, and cleaved backbone without 3′domain sequence was purified from gel. Seventy-five nanograms of the linear product containing the fragment and barcode was circularized using T4 DNA Ligase (NEB) 16°C overnight. The ligation product was treated with Lambda Exonuclease (NEB) and RecJF (NEB) for 16 h at 37°C (Balagurumoorthy et al. 2008 (link)). The remaining, circularized product was PCR amplified with primers containing Ion Torrent sites P1 & A. Concentration was determined by Bioanalyzer (Agilent) and sequenced on Ion Torrent using a 316 V2 chip (Thermo Fisher). Sequencing was conducted as 500-bp-long reads using an extended number of reagent flows in the 400 bp kit.
Protocol detail
Plasmid Barcode Sequencing Protocol
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Backbone
Bp 400
Chip
Digestion
Electrophoresis
Enzyme
Exonuclease
Library
Ligation
Plasmid
Primers
T4 dna ligase
Corresponding Organization :
Other organizations : Lund University
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Variable analysis
independent variables
- Restriction enzyme SalI used to digest the plasmid from the library
- T4 DNA Ligase used to circularize the linear product containing the fragment and barcode
- Lambda Exonuclease and RecJF enzymes used to treat the ligation product
- Digestion products separated by gel electrophoresis
- Cleaved backbone without 3' domain sequence purified from the gel
- Circularized product amplified by PCR with primers containing Ion Torrent sites P1 & A
- Sequencing of the PCR amplified product on Ion Torrent using a 316 V2 chip
- Incubation temperature of 16°C for the T4 DNA Ligase overnight ligation
- Incubation temperature of 37°C for 16 hours for the Lambda Exonuclease and RecJF treatment
- Positive control: None mentioned
- Negative control: None mentioned
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