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Team:Heidelberg/Notebook/Sensing Group/Notebook/2ndweek
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* PCR-purification of the all PCR-products eluted in 50 µl H<sub>2</sub>O | * PCR-purification of the all PCR-products eluted in 50 µl H<sub>2</sub>O | ||
* Miniprep of LuxQ no. 3 O/N culture and digestion with xbaI | * Miniprep of LuxQ no. 3 O/N culture and digestion with xbaI | ||
| - | * Fusion-PCR for Fusion-1 and | + | * Fusion-PCR for Fusion-1 and repetition 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 | + | * digestion of pDK48 plasmid and Fusion-1 frament with NcoI/NdeI (0.5 µl) and AgeI (1 µl) in NEBuffer 4 |
<div style="float: left;>[[Image:HD 080813-LuxQ PCR-pDK48 mini LuxQ3 xbaI.png|thumb|500px|Fusion-PCR for Fusion-1, LuxQ PCR, LuxQ-2 PCR, pDK48 miniprep and xbaI digestion of LuxQ no. 3]]</div> | <div style="float: left;>[[Image:HD 080813-LuxQ PCR-pDK48 mini LuxQ3 xbaI.png|thumb|500px|Fusion-PCR for Fusion-1, LuxQ PCR, LuxQ-2 PCR, pDK48 miniprep and xbaI digestion of LuxQ no. 3]]</div> | ||
<div style="float: left;">[[Image:HD 080813-F1 pDK48 Digestion.png|thumb|200px|Digestion of Fusion-1 and pDK48 with NcoI/NdeI/AgeI]]</div> | <div style="float: left;">[[Image:HD 080813-F1 pDK48 Digestion.png|thumb|200px|Digestion of Fusion-1 and pDK48 with NcoI/NdeI/AgeI]]</div> | ||
Revision as of 22:41, 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
- 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
- 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 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)
