Team:Biwako Nagahama/Original 3A Assembly

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]

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94℃　2min

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98℃　10sec

58℃   30sec             30cycles

68℃   2min30sec

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10℃　∞

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<Electrophoresis>

TE buffer・・・7μL

PCR Sample・・・2μL

10×Loading buffer・・・1μL

Total・・・10μL

0.7% Gel

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1 λ-HindⅢ            10μL

2 500bp DNA ladder  10μL

3 pSB1A3                10μL

4 pSB1K3                10μL

5 pSB1T3                10μL

6 pSB1C3                10μL

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<Exo Star process>