Team:Biwako Nagahama/Material & Method

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  • general protocol


    • Contents

      Material & Method

      Genaral protocol

      Distribution kit


      ↓With a pipette tip, punch a hole in the foil

      ↓Add 10μL of dH2O,and pipetting

      ↓Put 5min

      ↓Pipette 1uL of the resuspended DNA Transformation into your desired 100μL of competent cells

      ↓Hold on ice for 20min

      ↓Heat shock at 42℃ for 30sec

      ↓quickly

      ↓On ice for 2min

      ↓Add 900μL of SOCborth

      ↓Hold at 37℃ for 30min

      ↓Plating 100μL of DNA Transformation

      ↓Centrifuge for 1 min(13,000rpm)

      ↓Waste supernatant for 800μL, and pipetting

      ↓Plating all

      ↓Incubate at 37℃ (over night)


      Phenol-chloroform extraction


      Add Phenol-chloroform where is equivalent to Exo Star process sample

      ↓Centrifuge 4℃ 13,000rpm 5min

      Only supernatant was taken


      EtOH crystalization


      ↓Add 1μL 20mg/mL Glycogen

      ↓Mix

      ↓Add 1/10 volume 3M CH3COONa(pH5.2)

      ↓Add 2.5 times volume 99.5% EtOH

      ↓Vortex

      ↓Centrifuge 4℃ 13,000rpm 20min

      ↓Waste supernatant

      ↓Add 500μL 70% EtOH

      ↓Mix

      ↓Centrifuge for 10s in a table-top microcentrifuge

      ↓Waste supernatant

      ↓65℃ Dry up

      ↓Add 11μL TE buffer

      CelC

      Agro Notebook

      5/31 Cloning of CelC and Restriction Enzyme

      By Koki Tsutsumi

      CelC gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CelC gene has restriction enzyme sites,EcoRI and BamHI.

      Crds