"
Team:Biwako Nagahama/Material & Method
From 2013.igem.org
(→CelC) |
|||
| Line 64: | Line 64: | ||
== <h2>CelC</h2> == | == <h2>CelC</h2> == | ||
| + | <P></p> | ||
| + | <p>Agro Notebook</p> | ||
| + | <p>5/31 Cloning of CelC and Restriction Enzyme | ||
| + | </p> | ||
| + | <p>By Koki Tsutsumi | ||
| + | </p> | ||
| + | <p>CelC gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CelC gene has restriction enzyme sites,EcoRI and BamHI. </p> | ||
| + | |||
== <h2>Crds</h2> == | == <h2>Crds</h2> == | ||
Revision as of 17:43, 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
CelC
Agro Notebook
5/31 Cloning of CelC and Restriction Enzyme
By Koki Tsutsumi
CelC gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CelC gene has restriction enzyme sites,EcoRI and BamHI.
