Raw seq of phage EF_KTM
The flow cell was placed in the MinION sequencer pore check was performed by connecting the MinION to a computer and using the MinKNOW software. The functional pore count on a new flow cell should be >800. The kit components were thawed at room temperature, spinned down using a microcentrifuge andmixed by pipetting as indicated. Approximately ~400ng of DNA sample was added in a volume of 7.5 ul(diluting as necessary with nuclease free distilled water) to a PCR tube. 2.5ul of Fragmentation Mix (FRA) was added. To perform transposase-mediated fragmentation and adapter ligation, incubation was done. 1ul of Rapid Adapter (RAP) was added.The tube was then incubated for 5min at room temperature. The the library is stored on ice while preparing the flow cell. The Sequencing buffer (SQB) was thawed. Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature before placing the tubes on ice as soon as thawing is complete. The Sequencing Buffer and Flush Buffer were mixed. The lid of the MinION flow cell was opened and slide the flow cell's priming port cover clockwise so that the priming port is visible. Prepare the flow cell priming mix: add 30 l of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB) and mix by pipetting up and down. Load 800 l of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 minutes. Thoroughly mix the contents of the SQB and LB tubes by pipetting (the beads must not settle before adding to the subsequent library step). Gently lift the SpotON sample port cover on the MinION flow cell to make the SpotON sampleport accessible. Load 200 l of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles. Mix the prepared library gently by pipetting up and down just prior to loading. Add 75 l of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensureeach drop flows into the port before adding the next. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port,close the priming port and replace the MinION lid. Start the sequencing run, using the MinKNOW software interface.