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Team:Biwako Nagahama/Contact
From 2013.igem.org
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<p>______________________________________</p> | <p>______________________________________</p> | ||
<p>1 λ-HindⅢ 10</p> | <p>1 λ-HindⅢ 10</p> | ||
| - | <p>2 500bp DNA ladder 10</p> | + | <p>2 500bp DNA ladder 10</p> |
<p>3 pSB1A3 10</p> | <p>3 pSB1A3 10</p> | ||
<p>4 pSB1K3 10</p> | <p>4 pSB1K3 10</p> | ||
<p>5 pSB1T3 10</p> | <p>5 pSB1T3 10</p> | ||
<p>6 pSB1C3 10</p> | <p>6 pSB1C3 10</p> | ||
| - | + | <p>______________________________________</p> | |
<div class="spacer"></div> | <div class="spacer"></div> | ||
Revision as of 12:44, 27 September 2013
Contents |
3A Assembly Biwako-Nagahama original protocol
Reason
We succeeded in 3A Assembly using Linear Backbone from the Distribution Kit obtained from the iGEM Headquarter. But we could not succeeded in making 3A Assembly of the linear backbone taking reference of the Protocol of linear backbone found in the iGEM Homepage. So, we made our own 3A Assembly protocol suitable to our own lab environment that could increase the success rate of the experiment.
On discussing about the 3A Assembly with other participating teams, we found that other teams were not preparing their own protocol regarding 3A Assembly. We found that the 3A Assembly could be performed easily in the environment with certain restrictions. Because 3A Assembly is Assmbly method of the high probability not to need PCR and gel purification. So we tried to debug the available protocols and make our own protocols suitable to our own experimenting environment and help other teams with similar environment condition.
“Protocol”
<iGEM Backbone(pSB1C3,pSB1K3,pSB11A3,pSB1T3) manufacture>
2×KODFx buffer・・・25μL
dNTPs[2mM]・・・0μL
[http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones ※1] SB-prep-3P-1・・・1.5μL
[http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones ※2] SB-prep-2Ea・・・1.5μL
KODFx[1.0U/μL]・・・1.0μL
MilliQ H2O・・・10.0μL
Template[1～10ng/μL](pSB1C3 or pSB1K3 or pSB1T3 or pSB1A3)・・・1.0μL
Total・・・50.0μL
[http://www.toyobo-global.com/seihin/xr/lifescience/products/pcr_002.html ToYoBo KOD FX]
↓
94℃ 2min
↓
__________________
98℃ 10sec
58℃ 30sec 30cycles
68℃ 2min30sec
__________________
↓
10℃ ∞
↓
<Electrophoresis>
TE buffer・・・7μL
PCR Sample・・・2μL
10×Loading buffer・・・1μL
Total・・・10μL
0.7% Gel
______________________________________
1 λ-HindⅢ 10
2 500bp DNA ladder 10
3 pSB1A3 10
4 pSB1K3 10
5 pSB1T3 10
6 pSB1C3 10
______________________________________
