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Team:Heidelberg/Notebook/Sensing Group/Notebook/2ndweek
From 2008.igem.org
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* digestion of Fusion-1 with AgeI in NEBuffer 1 | * digestion of Fusion-1 with AgeI in NEBuffer 1 | ||
* gel extraction of sequentially digested Fusion-1 and pDK48 products | * gel extraction of sequentially digested Fusion-1 and pDK48 products | ||
| - | * new digestion of Fusion-1 with NcoI/NdeI and gel extraction, because previous digestion did not yield expected bands (there should be two small bands at 516bp and 392bp) | + | * new digestion of Fusion-1 with NcoI/NdeI (NEBuffer 4) and gel extraction, because previous digestion did not yield expected bands (there should be two small bands at 516bp and 392bp) |
[[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.]] | ||
Revision as of 23:34, 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 (NEBuffer 3 + BSA) --> negative result
- PCR for LuxQ, LuxQ-1, LuxQ-2, Tar-1, Tar-2 with Taq Mastermix
- 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
- preparation of O/N culture for LuxQ no. 3
Wednesday, 08/13/2008
- PCR-purification of the all PCR-products eluted in 50 µl H2O
- Miniprep of LuxQ no. 3 O/N culture and digestion with xbaI (NEBuffer 2 + BSA)
- Fusion-PCR for Fusion-1 and repetition of PCR for LuxQ-2 and LuxQ
- digestion of pDK48 plasmid and Fusion-1 frament with NcoI/NdeI (0.5 µl) and AgeI (1 µl) in NEBuffer 4
Thursday, 08/14/2008
- Gel for 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
- gel extraction of sequentially digested Fusion-1 and pDK48 products
- new digestion of Fusion-1 with NcoI/NdeI (NEBuffer 4) and gel extraction, because previous digestion did not yield expected bands (there should be two small bands at 516bp and 392bp)
- Ligation of 5 µl Fusion-1 with 5 µl pDK48 (both digested with NcoI/NdeI )
- Transformation into DH5alpha (5 µl Ligation products)
