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Team:Evry/Notebook/Transposons/14.10.14
From 2014.igem.org
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<h4> Ligation of BBa_K1413001 </h4> | <h4> Ligation of BBa_K1413001 </h4> | ||
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| - | strategy: | + | strategy:</p> |
| + | <br><div align="center"> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/9/95/Evry_Strat2.jpg" width="50%"/></div> | ||
<br> | <br> | ||
| - | < | + | <p> |
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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 | ||
| - | <br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution | + | <br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution</p> |
<br><br> | <br><br> | ||
| - | + | <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 | ||
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50ul electrocompetent cells | 50ul electrocompetent cells | ||
2 ul Ligation product of I° strategy / II° strategy in <i>E.coli DH5alpha</i>. | 2 ul Ligation product of I° strategy / II° strategy in <i>E.coli DH5alpha</i>. | ||
| - | <br> | + | <br></p> |
Revision as of 22:42, 17 October 2014
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.
