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Team:Evry/Notebook/Transposons/14.10.14
From 2014.igem.org
(Difference between revisions)
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| - | + | <h4> Ligation of BBa_K1413001 </h4> | |
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<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 | ||
| - | + | <br> | |
Ligation: | Ligation: | ||
| - | I° strategy: | + | <br>I° strategy: |
<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 | <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 | ||
| - | + | <br><br> | |
II° Strategy: | II° Strategy: | ||
<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 | <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 | ||
| - | + | <br><br> | |
Clean up: | Clean up: | ||
| - | II° strategy: | + | <br>II° strategy: |
| - | Ligation product by Thermoscientific "PCR clean up" Kit in 30 ul final elution | + | <br>Ligation product by Thermoscientific "PCR clean up" Kit in 30 ul final elution |
| - | + | <br><br> | |
Digestion: II° strategy | Digestion: II° strategy | ||
Revision as of 22:05, 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. 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.
Oct 14
