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Team:Biwako Nagahama/Material & Method
From 2013.igem.org
(→Genaral protocol) |
(→Genaral protocol) |
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<p>↓Plating all</p> | <p>↓Plating all</p> | ||
<p>↓Incubate at 37℃ (over night)</p> | <p>↓Incubate at 37℃ (over night)</p> | ||
| + | ---- | ||
| + | <h3>Phenol-chloroform extraction</h3> | ||
| + | ---- | ||
| + | <p>Add Phenol-chloroform where is equivalent to Exo Star process sample</p> | ||
| + | <p>↓Centrifuge 4℃ 13,000rpm 5min</p> | ||
| + | <p>Only supernatant was taken</p> | ||
| + | <p>↓</p> | ||
| + | <h3>EtOH crystalization</h3> | ||
| + | <p>Add 1μL 20mg/mL Glycogen</p> | ||
| + | <p>↓Mix</p> | ||
| + | <p>Add 1/10 volume 3M CH3COONa(pH5.2)</p> | ||
| + | <p>↓</p> | ||
| + | <p>Add 2.5 times volume 99.5% EtOH</p> | ||
| + | <p>↓Vortex</p> | ||
| + | <p>↓Centrifuge 4℃ 13,000rpm 20min</p> | ||
| + | <p>Waste supernatant</p> | ||
| + | <p>↓</p> | ||
| + | <p>Add 500μL 70% EtOH</p> | ||
| + | <p>↓Mix</p> | ||
| + | <p>↓Centrifuge for 10s in a table-top microcentrifuge</p> | ||
| + | <p>Waste supernatant</p> | ||
| + | <p>↓</p> | ||
| + | <p>65℃ Dry up</p> | ||
| + | <p>↓</p> | ||
| + | <p>Add 11μL TE buffer</p> | ||
== <h2>CelC</h2> == | == <h2>CelC</h2> == | ||
Revision as of 17:34, 27 September 2013
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
