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Team:Heidelberg/Notebook/Sensing Group/Notebook/2ndweek
From 2008.igem.org
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[[Image:HD 080815-F1 digestion pDK48.png|left|thumb|250px|Gelextraction of digested Fusion-1 and purified PCR product as well as pDK48 digested. Expected bands at 516bp and 392bp are not visible.]] | [[Image:HD 080815-F1 digestion pDK48.png|left|thumb|250px|Gelextraction of digested Fusion-1 and purified PCR product as well as pDK48 digested. Expected bands at 516bp and 392bp are not visible.]] | ||
[[Image:HD 080815-F1 digestion NcoI NdeI.png|left|thumb|200px|Fusion-1 digested with NcoI/NdeI. Expected bands of 1025bp and 908bp had to be cut out together. Wanted band is the longer one.]] | [[Image:HD 080815-F1 digestion NcoI NdeI.png|left|thumb|200px|Fusion-1 digested with NcoI/NdeI. Expected bands of 1025bp and 908bp had to be cut out together. Wanted band is the longer one.]] | ||
| + | * Ligation of 5 µl Fusion-1, digested with NcoI/NdeI and 5 µl pDK48 | ||
| + | * Transformation into DH5a (5 µl Ligation & 15 µl Ligation) | ||
<br style="clear: both;"/> | <br style="clear: both;"/> | ||
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/3rdweek | Go to 3rd week]] | [[Team:Heidelberg/Notebook/Sensing_Group/Notebook/3rdweek | Go to 3rd week]] | ||
Revision as of 13:43, 26 October 2008
Contents |
Monday, 08/11/2008
- preparation of LuxS harboring cells O/N culture
Tuesday, 08/12/2008
- Miniprep of correctly sequenced LuxS and Glycerol-stock
- LuxQ no. 3 checked via digestion with NcoI/BamHI --> negative result
- PCR for LuxQ, LuxQ-1, LuxQ-2, Tar-1, Tar-2 with Taq Mastermix
- preparation of O/N culture for LuxQ no. 3
Wednesday, 08/13/2008
- PCR-purification of the 5 PCR-products eluted in 50 µl H2O
- Miniprep of LuxQ no. 3 O/N culture and digestion with xbaI
- Fusion-PCR for Fusion-1 and repetetion of PCR for LuxQ-2 and LuxQ
- digestion of pDK48 and Fusion-1 with NcoI/NdeI (0.5 µl) and AgeI (1 µl) in NEBuffer 4
Thursday, 08/14/2008
- Gel from LuxQ, LuxQ2
- sequentiell digestion of Fusion-1 with NcoI and NdeI (NEBuffer 3)
- Double digestion of pDK48 with NcoI/NdeI (NEBuffer 4)
Friday, 08/15/2008
- digestion of Fusion-1 with AgeI in NEBuffer 1
- Gelextraction of sequentially digested Fusion-1 and pDK48
- new digestion of Fusion-1 with NcoI/NdeI and Gelextraction, because previous digestion did not yield expected bands (there should be two bands at 516bp and 392bp)
- Ligation of 5 µl Fusion-1, digested with NcoI/NdeI and 5 µl pDK48
- Transformation into DH5a (5 µl Ligation & 15 µl Ligation)
