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Team:Biwako Nagahama/general protocol
From 2013.igem.org
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
↓
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
