Team:Evry/Notebook/Transposons/14.10.14

From 2014.igem.org

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== Ligation of BBa_K1413001==
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<h4> Ligation of BBa_K1413001 </h4>
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strategy:
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strategy:</p>
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<img src="https://static.igem.org/mediawiki/2014/9/95/Evry_Strat2.jpg" width="50%"/></div>
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<img src="" width="50%"/>
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Digestion of BBa_K1413001 by E/S :
Digestion of BBa_K1413001 by E/S :
<br>50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (35 ng/ul) BBa_1413001 in pSB1C3, 1 ul E/S, 39 ul H2O. 1h
<br>50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (35 ng/ul) BBa_1413001 in pSB1C3, 1 ul E/S, 39 ul H2O. 1h
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<br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution
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<br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution</p>
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<br>
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<br><br>
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<p>
Digestion of Kan 82/83 by E/S & X/P:
Digestion of Kan 82/83 by E/S & X/P:
<br>50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul (30 ng/ul) Kan 82/83 in pSB1C3, 1 ul E/S or X/P, 38ul H2O. 1h
<br>50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul (30 ng/ul) Kan 82/83 in pSB1C3, 1 ul E/S or X/P, 38ul H2O. 1h
<br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution
<br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution
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<br><br>
Ligation:  
Ligation:  
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I° strategy:
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<br>I° strategy:
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<br> 20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 3 ul Kan 82/83 + 5 BBa_1413044 transposon plasmid. 4 ul H2O. 1h
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<br> 20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 3 ul Kan 82/83 + 5 BBa_1413044 transposon plasmid. 4 ul H2O. 1 ul T4 ligase. 1h
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<br><br>
II° Strategy:
II° Strategy:
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<br> 20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 5 BBa_1413044 transposon plasmid. 4 ul H2O. 1h
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<br> 20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 5 BBa_1413044 transposon plasmid. 4 ul H2O.1 ul T4 ligase. 1h
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<br><br>
Clean up:
Clean up:
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II° strategy:
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<br>II° strategy:
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Ligation product by Thermoscientific "PCR clean up" Kit in 30 ul final elution
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<br>Ligation product by Thermoscientific "PCR clean up" Kit in 30 ul final elution
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<br><br>
Digestion: II° strategy
Digestion: II° strategy
<br> 50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul ligation product, 1 ul E/X, 39 ul H2O. 1h
<br> 50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul ligation product, 1 ul E/X, 39 ul H2O. 1h
<br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution
<br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution
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<br>
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<br><br>
Ligation:
Ligation:
<br>
<br>
II° Strategy:
II° Strategy:
<br> 20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul ligation product E/X + 5 ul Kan 82/83 E/S. 4 ul H2O. 1h
<br> 20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul ligation product E/X + 5 ul Kan 82/83 E/S. 4 ul H2O. 1h
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<br>
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<br><br>
Transformation: electrocompetent
Transformation: electrocompetent
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<br><br>
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Ligation product of I° strategy / II° strategy in <i>E.coli DH5alpha</i></i>.
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50ul electrocompetent cells
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2 ul Ligation product of I° strategy / II° strategy in <i>E.coli DH5alpha</i>.
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<br></p>
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</p>
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<span class="cd-date">Oct 14</span>  
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<span class="cd-date">Oct 13</span>  
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Latest revision as of 22:55, 17 October 2014

Picture

Ligation of BBa_K1413001


strategy:



Digestion of BBa_K1413001 by E/S :
50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (35 ng/ul) BBa_1413001 in pSB1C3, 1 ul E/S, 39 ul H2O. 1h
Then Thermoscientific "PCR clean up" Kit in 30 ul final elution



Digestion of Kan 82/83 by E/S & X/P:
50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul (30 ng/ul) Kan 82/83 in pSB1C3, 1 ul E/S or X/P, 38ul H2O. 1h
Then Thermoscientific "PCR clean up" Kit in 30 ul final elution

Ligation:
I° strategy:
20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 3 ul Kan 82/83 + 5 BBa_1413044 transposon plasmid. 4 ul H2O. 1 ul T4 ligase. 1h

II° Strategy:
20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 5 BBa_1413044 transposon plasmid. 4 ul H2O.1 ul T4 ligase. 1h

Clean up:
II° strategy:
Ligation product by Thermoscientific "PCR clean up" Kit in 30 ul final elution

Digestion: II° strategy
50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul ligation product, 1 ul E/X, 39 ul H2O. 1h
Then Thermoscientific "PCR clean up" Kit in 30 ul final elution

Ligation:
II° Strategy:
20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul ligation product E/X + 5 ul Kan 82/83 E/S. 4 ul H2O. 1h

Transformation: electrocompetent

50ul electrocompetent cells 2 ul Ligation product of I° strategy / II° strategy in E.coli DH5alpha.

Oct 14

Retrieved from "http://2014.igem.org/Team:Evry/Notebook/Transposons/14.10.14"