Team:Evry/Notebook/Transposons/14.10.14

From 2014.igem.org

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<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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Ligation:
Ligation:
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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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Transformation:
 
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== Obtaining the Kan gene on pNK2 ==
 
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PCR: enzyme: Q5; template: 150 ng pNK2; oligo: F 82/ R 83; Tm tested: 55/60/65; elongation time: 1m30s
 
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<img src="" width="50%"/>
 
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Purification sur gel:
 
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Kit thermoscientific "Gel extraction". Elution final in 30 ul.
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Transformation: electrocompetent
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Ligation product of I° strategy / II° strategy in <i>E.coli DH5alpha</i> & <i>Pseudovibrio denitrificans</i>.
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Revision as of 20:21, 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. 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. 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
Ligation product of I° strategy / II° strategy in E.coli DH5alpha & Pseudovibrio denitrificans.

Oct 13