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Team:Evry/Notebook/Transposons/16.10.14
From 2014.igem.org
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<h4> Show BBa_1413044 work even empty in DH5alpha </h4> | <h4> Show BBa_1413044 work even empty in DH5alpha </h4> | ||
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8 colonies of DH5alpha were transformed with BBa_1413044 and grown in 3 ml LB + 2 negative control | 8 colonies of DH5alpha were transformed with BBa_1413044 and grown in 3 ml LB + 2 negative control | ||
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PCR: enzyme: Q5; template: 3 ul of supernatent for the culture; oligo: F 100/ R 101; Tm tested: 55; elongation time: 1m00s | PCR: enzyme: Q5; template: 3 ul of supernatent for the culture; oligo: F 100/ R 101; Tm tested: 55; elongation time: 1m00s | ||
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| - | <img src="https://static.igem.org/mediawiki/2014/b/b9/Evry_Is_ecoli.JPG" width="50%"/> | + | <div align="center"> |
| + | <img src="https://static.igem.org/mediawiki/2014/b/b9/Evry_Is_ecoli.JPG" width="50%"/></div> | ||
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<h4> defining where the transposase integrate </h4> | <h4> defining where the transposase integrate </h4> | ||
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find enzyme with a good cut numbers in the genome and not in our insert: HindIII | find enzyme with a good cut numbers in the genome and not in our insert: HindIII | ||
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<br><br>then PCR | <br><br>then PCR | ||
<br>PCR: enzyme: Q5; template: 3 ul of digestion by XbaI; oligo: F 105/ R 106; Tm tested: 55; elongation time: 4m00s | <br>PCR: enzyme: Q5; template: 3 ul of digestion by XbaI; oligo: F 105/ R 106; Tm tested: 55; elongation time: 4m00s | ||
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| - | <img src="https://static.igem.org/mediawiki/2014/a/af/Evry_Genome_cut_religated.JPG" width="50%"/> | + | <div align="center"> |
| + | <img src="https://static.igem.org/mediawiki/2014/a/af/Evry_Genome_cut_religated.JPG" width="50%"/></div> | ||
</p> | </p> | ||
Revision as of 22:49, 17 October 2014
Show BBa_1413044 work even empty in DH5alpha
8 colonies of DH5alpha were transformed with BBa_1413044 and grown in 3 ml LB + 2 negative control
Preparation for PCR colony:
Spin down 500 ul culture at 14000 g - 2 min, resuspend in 100 ul H20 MilliQ, put at 95 C° for 10 min then spin down at 14000 g - 2 min.
PCR: enzyme: Q5; template: 3 ul of supernatent for the culture; oligo: F 100/ R 101; Tm tested: 55; elongation time: 1m00s
defining where the transposase integrate
find enzyme with a good cut numbers in the genome and not in our insert: HindIII
Digestion of 8 Pseudovibrio denitrificans DNA preparation by HindIII
50ul final volume, 200 ng DNA preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul HindIII. 1h
Then religate:
Ligation: 20 ul final volume, 2 ul 10x T4 ligase buffer, 1 ul T4
then light digestion with XbaI
50ul final volume, 20 ul DNA religated preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul XbaI enzyme. 15 min
deactivation at 80 C° for 20 min
then PCR
PCR: enzyme: Q5; template: 3 ul of digestion by XbaI; oligo: F 105/ R 106; Tm tested: 55; elongation time: 4m00s
