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Team:Evry/Notebook/Transposons/13.10.14
From 2014.igem.org
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| + | <h4> BBa_1413044 retrieval of the transposon plasmid </h4> | ||
| + | <br><p> | ||
| + | strategy:</p> | ||
| + | <div align="center"> | ||
| + | <br><img src="https://static.igem.org/mediawiki/2014/3/33/Evry_Strat1.jpg" width="75%"/></div> | ||
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Digestion of BBa_1413044 by BglII : | Digestion of BBa_1413044 by BglII : | ||
<br>50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (20 ng/ul) BBa_1413044 in pSB1C3, 1 ul BglII, 39 ul H2O. | <br>50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (20 ng/ul) BBa_1413044 in pSB1C3, 1 ul BglII, 39 ul H2O. | ||
<br> | <br> | ||
Gel electrophoresis: | Gel electrophoresis: | ||
| - | <br> | + | <br><div align="center"> |
| - | <img src="https://static.igem.org/mediawiki/2014/3/31/Evry_Bba_transpo.JPG" width=" | + | <img src="https://static.igem.org/mediawiki/2014/3/31/Evry_Bba_transpo.JPG" width="25%"/></div> |
<br> | <br> | ||
Purification sur gel: | Purification sur gel: | ||
<br> | <br> | ||
Kit thermoscientific "Gel extraction". Elution final in 30 ul. | Kit thermoscientific "Gel extraction". Elution final in 30 ul. | ||
| - | <br> | + | <br></p> |
| - | + | <h4>Obtaining the Kan gene on pNK2</h4> | |
| - | <br> | + | <br><p> |
| - | PCR: enzyme: Q5; template: 150 ng pNK2; oligo: F 82/ R 83; Tm tested: 55/60/65; elongation time: 1m30s | + | PCR: enzyme: Q5; template: 150 ng pNK2 first three well from new stock, next ones the old stock; oligo: F 82/ R 83; Tm tested: 55/60/65; elongation time: 1m30s |
| - | <br> | + | <br></p><div align="center"> |
| - | <img src="https://static.igem.org/mediawiki/2014/9/90/Evry_Kan_extract.JPG" width=" | + | <img src="https://static.igem.org/mediawiki/2014/9/90/Evry_Kan_extract.JPG" width="40%"/></div> |
| - | <br> | + | <br><p> |
Purification sur gel: | Purification sur gel: | ||
<br> | <br> | ||
Kit thermoscientific "Gel extraction". Elution final in 30 ul. | Kit thermoscientific "Gel extraction". Elution final in 30 ul. | ||
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Latest revision as of 22:41, 17 October 2014
BBa_1413044 retrieval of the transposon plasmid
strategy:
Digestion of BBa_1413044 by BglII :
50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (20 ng/ul) BBa_1413044 in pSB1C3, 1 ul BglII, 39 ul H2O.
Gel electrophoresis:
Purification sur gel:
Kit thermoscientific "Gel extraction". Elution final in 30 ul.
Obtaining the Kan gene on pNK2
PCR: enzyme: Q5; template: 150 ng pNK2 first three well from new stock, next ones the old stock; oligo: F 82/ R 83; Tm tested: 55/60/65; elongation time: 1m30s
Purification sur gel:
Kit thermoscientific "Gel extraction". Elution final in 30 ul.
