Team:Biwako Nagahama/Material & Method

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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

Crds