Team:Biwako Nagahama/Contact

From 2013.igem.org

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== <h2>3A Assembly Biwako-Nagahama original protocol</h2> ==
 
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<h3>'''Reason'''</h3>
 
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<p></p>
 
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<p>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.
 
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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.
 
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<h5>Team Contact</h5>
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== “Protocol” ==
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[http://www.nagahama-i-bio.ac.jp/ Nagahama Institute of Bio Science and Technology]
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<h3><iGEM Backbone(pSB1C3,pSB1K3,pSB11A3,pSB1T3) manufacture></h3>
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<p>2×KODFx buffer・・・25μL</p>
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Nagahama city,Shiga Prefecture,Japan
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<p>dNTPs[2mM]・・・0μL</p>
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<p>[http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones ※1] SB-prep-3P-1・・・1.5μL</p>
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igem.biwako@gmail.com
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<p>[http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones ※2] SB-prep-2Ea・・・1.5μL</p>
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<p>KODFx[1.0U/μL]・・・1.0μL</p>
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[https://www.facebook.com/IgemBiwako?ref=hl/ Facebook Team page]
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<p>MilliQ H2O・・・10.0μL</p>
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<p>Template[1~10ng/μL](pSB1C3 or pSB1K3 or pSB1T3 or pSB1A3)・・・1.0μL</p>
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<p>Total・・・50.0μL</p>
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[http://www.toyobo-global.com/seihin/xr/lifescience/products/pcr_002.html ToYoBo KOD FX]
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<p>↓</p>
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<p>94℃ 2min</p>
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<p> ↓</p>
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<p>__________________</p>
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<p>98℃ 10sec</p>   
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<p>58℃ &nbsp; 30sec &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;      30cycles</p>
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<p>68℃ &nbsp; 2min30sec</p>
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<p>__________________</p>
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<p> ↓</p>
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<p>10℃ ∞</p>
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<h3><Electrophoresis></h3>
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<p>TE buffer・・・7μL</p>
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<p>PCR Sample・・・2μL</p>
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<p>10×Loading buffer・・・1μL</p>
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<p>Total・・・10μL</p>
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<p>0.7% Gel</p>
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<p>No.&nbsp;Sample nameTotal</p>
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<p>______________________________________</p>
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<p>1&nbsp;λ-HindⅢ&nbsp;&nbsp;10</p>
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<p>2&nbsp;500bp DNA ladder&nbsp;&nbsp;&nbsp;&nbsp;10</p>
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<p>3&nbsp;pSB1A3&nbsp;&nbsp;&nbsp;&nbsp;10</p>
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<p>4&nbsp;pSB1K3&nbsp;&nbsp;&nbsp;&nbsp;10</p>
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<p>5&nbsp;pSB1T3&nbsp;&nbsp;&nbsp;&nbsp;10</p>
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<p>6&nbsp;pSB1C3&nbsp;&nbsp;&nbsp;&nbsp;10</p>
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Latest revision as of 12:50, 27 September 2013


Team Contact

Nagahama Institute of Bio Science and Technology

Nagahama city,Shiga Prefecture,Japan

igem.biwako@gmail.com