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Team:Biwako Nagahama/general protocol
From 2013.igem.org
Contents |
Genaral protocol
BIOTAQ™ DNA PolymerasePCR
| dH2O | 12.7μL |
| 10×NH4 buffer | 2.5μL |
| 50mM MgCl2 | 0.75μL |
| 25mM dNTPs | 2μL |
| 10μM F Primer | 1μL |
| 10μM R Primer | 1μL |
| BIO Taq(5U/μL) | 0.05μL |
| Template(<500ng) | 5μL |
| Total | 25μL |
95℃ 30sec
↓
95℃ 10sec
55℃ 20sec 30cycles
72℃ 2min
↓
72℃ 2min
↓
10℃ ∞
[http://www.toyobo-global.com/seihin/xr/lifescience/products/pcr_002.html TOYOBO KOD Fx]PCR
| 2×KODFx buffer | 25μL |
| dNTPs[2mM] | 10μL |
| 10μM F Primer | 1.5μL |
| 10μM R Primer | 1.5μL |
| KOD Fx[1.0U/μL] | 1.0μL |
| MilliQ H2O | 10.0μL |
| Template(<500ng) | 1.0μL |
| Total | 50μL |
94℃ 2min
↓
98℃ 10sec
58℃ 30sec 30cycles
68℃ 2min30sec
↓
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
By. Syohei Takeshita
