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Team:Heidelberg/Notebook/Sensing Group/Notebook/1stweek
From 2008.igem.org
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=== Cloning of LuxP === | === Cloning of LuxP === | ||
| - | [[Image:HD_080805-LuxP_PCR.png | + | [[Image:HD_080805-LuxP_PCR.png|thumb|150px|LuxP colony PCR]] |
| - | [[Image:HD 080805-LuxQ S PCR.png|right|thumb|upright|LuxQ, LuxS PCR]] | + | <div style="float: right; clear:none">[[Image:HD 080805-LuxQ S PCR.png|right|thumb|upright|LuxQ, LuxS PCR]]</div> |
* PCR of LuxP from ''V. harveyi'' colony | * PCR of LuxP from ''V. harveyi'' colony | ||
* Gel Purification of LuxP eluted in 30 µl H<sub>2</sub>O | * Gel Purification of LuxP eluted in 30 µl H<sub>2</sub>O | ||
Revision as of 10:07, 26 October 2008
Contents |
Monday, 08/04/2008
Preparations
Preparations were done at the end of the week before
- Transformation of pDK48 and pTrc99a in E. coli DH5a (0.5 µl plasmid-DNA + 50 µl competent cells)
- Glycerol-stock of Vibrio harveyi BB120, BB886, mm30, BB178, BB125
- picked pTrc99a and pDK48 cultures and inoculated in 3 ml LB overnight
- V. harveyi cultured on LB+-Agar plate
- MiniPrep of pTrc99a and pDK48 from DHha
Cloning of LuxP
- PCR of LuxP from V. harveyi colony
- Gel Purification of LuxP eluted in 30 µl H2O
- Digestion of LuxP with SalI and NotI of pDK48 and LuxP-Gel-Purification product: 2 µl NEB-Buffer 3 + 0.5 µl SalI/NotI + 10 µl DNA + 7 µl H2O
- Ligation with vector:insert ratio 1:3 and 1:1
Cloning of LuxS
- PCR of V. harveyi genome and PCR purification
- digestion of LuxS and pTrc99a with BamHI/NcoI
- Ligation with vector:insert ratio 1:3 and 1:1
