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Team:Evry/Notebook/Transposons/16.10.14
From 2014.igem.org
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== Show BBa_1413044 work even empty in DH5alpha == | == Show BBa_1413044 work even empty in DH5alpha == | ||
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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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Preparation for PCR colony: | 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. | + | <br>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. |
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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%"/> | <img src="https://static.igem.org/mediawiki/2014/b/b9/Evry_Is_ecoli.JPG" width="50%"/> | ||
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== defining where the transposase integrate == | == defining where the transposase integrate == | ||
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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 | ||
| - | Then religate with T4 | + | <br><br>Then religate with T4 |
| - | then light digestion with XbaI | + | <br><br>then light digestion with XbaI |
| - | then PCR | + | <br><br>then PCR |
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<img src="https://static.igem.org/mediawiki/2014/a/af/Evry_Genome_cut_religated.JPG" width="50%"/> | <img src="https://static.igem.org/mediawiki/2014/a/af/Evry_Genome_cut_religated.JPG" width="50%"/> | ||
Revision as of 22:12, 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
Then religate with T4
then light digestion with XbaI
then PCR
