Team:Biwako Nagahama/general protocol

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Contents

Genaral protocol

BIOTAQ™ DNA PolymerasePCR


dH2O12.7μL
10×NH4 buffer2.5μL
50mM MgCl20.75μL
25mM dNTPs2μL
10μM F Primer1μL
10μM R Primer1μL
BIO Taq(5U/μL)0.05μL
Template(<500ng)5μL
Total25μL

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 buffer25μL
dNTPs[2mM]10μL
10μM F Primer1.5μL
10μM R Primer1.5μL
KOD Fx[1.0U/μL]1.0μL
MilliQ H2O10.0μL
Template(<500ng)1.0μL
Total50μL

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