Team:Biwako Nagahama/Material & Method

From 2013.igem.org

(Difference between revisions)
Line 13: Line 13:
</html> ==
</html> ==
-
== <h2>Genaral protocol</h2> ==
 
-
<h3>Distribution kit</h3>
 
-
----
 
-
<p>↓With a pipette tip, punch a hole in the foil</p>
 
-
<p>↓Add 10μL of dH2O,and pipetting</p>
 
-
<p>↓Put 5min</p>
 
-
<p>↓Pipette 1uL of the resuspended DNA Transformation into your desired 100μL of competent cells</p>
 
-
<p>↓Hold on ice for 20min</p>
 
-
<p>↓Heat shock at 42℃ for 30sec</p>
 
-
<p>↓quickly</p>
 
-
<p>↓On ice for 2min</p>
 
-
<p>↓Add 900μL of SOCborth</p>
 
-
<p>↓Hold at 37℃ for 30min</p>
 
-
<p>↓Plating 100μL of DNA Transformation</p>
 
-
<p>↓Centrifuge for 1 min(13,000rpm)</p>
 
-
<p>↓Waste supernatant for 800μL, and pipetting</p>
 
-
<p>↓Plating all</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>
 
-
----
 
-
<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>↓Add 2.5 times volume 99.5% EtOH</p>
 
-
<p>↓Vortex</p>
 
-
<p>↓Centrifuge 4℃ 13,000rpm 20min</p>
 
-
<p>↓Waste supernatant</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>↓65℃ Dry up</p>
 
-
<p>↓Add 11μL TE buffer</p>
 
== <h2>CelC</h2> ==
== <h2>CelC</h2> ==

Revision as of 18:01, 27 September 2013

Contents

Material & Method

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.

Biwako-Nagahama T.Ksenpai celC1.png

Crds