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