"
Team:Heidelberg/Notebook/Sensing Group/Notebook/6thweek
From 2008.igem.org
(Difference between revisions)
(→Fusion chimeras) |
(→Fusion chimeras) |
||
| Line 163: | Line 163: | ||
* Two Fusion PCR (30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)) with PCR purification. | * Two Fusion PCR (30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)) with PCR purification. | ||
* Gel Extraction of LuxQ 1+2, Tar 1+2 | * Gel Extraction of LuxQ 1+2, Tar 1+2 | ||
| - | [[Image:HD 080910-F1 2 Phusion2.png|left|thumb| | + | [[Image:HD 080910-F1 2 Phusion2.png|left|thumb|200px|First Fusion PCR]][[Image:HD 080910-Fusion PCR 2.png|left|thumb|400px|another fusion PCR]] |
<br style="clear: both;" /> | <br style="clear: both;" /> | ||
Revision as of 17:21, 26 October 2008
Contents |
Monday, 09/08/2008
- Liquid Culture of 5 colonies from LuxQ Transformation and 1 from F1 transformation
- Miniprep of Liquid Culture from previous Transformation (done by Chenchen). Incubated for two days @ RT and about 6 h @ 37 °C
- Digestion of the Miniprep products with xbaI (Buffer2 + BSA, 1 ul enzyme @ 37 °C about 40min)
Sequencing @ GATC yielded correct sequence for LuxQ no. 1
- PCR for LuxQ and LuxP with Phusion (25 µl Mastermix, 0.5 µl each Primer, 0(1) µl DMSO, 24(23) µl H20, 0.5 µl Template)
- PCR Programme: 5 min @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C | 30 s @ 72 °C || 5 min @ 72 °C | 4 °C
Tuesdsay, 09/09/2008
- Miniprep of LuxQ colonies no. 1,2,3,5,6
- PCR for LuxQ fragment 1 and 2, Tar fragment 1 and 2 for Phusion receptor with Phusion-Polymerase
- PCR Programme: 30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)
- Fusion-PCR for Fusion-1 and Fusion-2 constructs
- O/N Culture of 10 LuxP in pDK48 colonies
Wednesday, 09/10/2008
LuxP cloning
- Miniprep of O/N LuxP cultures and digestion with NcoI (2h @ 37 °C)
Fusion chimeras
- PCR for LuxQ 1+2, Tar 1+2 and Gel Extraction to get rid of first primers
- Fusion-PCR of LuxQ1+Tar1 und LuxQ2+Tar2 with Phusion and 55 °C annealing --> No product
- PCR of LuxQ fragment 1b, LuxQ fragment 1c, LuxQ fragment 2b, LuxQ fragment 2c, Tar fragment 1b, Tar fragment 1c, Tar fragment 2b, Tar fragment 2c with Phusion and 55 °C annealing. Products were purified via Gelextraction to get rid of first primers
- Two Fusion PCR (30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)) with PCR purification.
- Gel Extraction of LuxQ 1+2, Tar 1+2
