Team:Biwako Nagahama/general protocol

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Contents

Genaral protocol

<Colony PCR>(check Trance formation)

Take a single colony from the plate and then mix it with 30μL of de-ionized water(dH2O)

dH2O・・・12.7μL

10×NH4 buffer・・・2.5μL

50mM MgCl2・・・0.75μL

25mM dNTPs・・・2μL

10μM [http://parts.igem.org/Part:BBa_G1004 ※3] BBa_G1004・・・1μL

10μM [http://parts.igem.org/Part:BBa_G1005 ※4] BBa_G1005・・・1μL

BIO Taq(5U/μL)・・・0.05μL

Template(dH2O mixed colony)・・・5μL

Total・・・25μL

BIOTAQ™ DNA Polymerase


95℃ 30sec

95℃ 10sec

55℃ 20sec 30cycles

72℃ 2min

72℃ 2min

10℃ ∞



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