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Team:Heidelberg/Notebook/Sensing Group/Notebook/4thweek
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* test digestion of pDK48 with XbaI (NEBuffer 2 + BSA) and PstI (NeBuffer 3 + BSA) to check wehther the vector is the right one | * test digestion of pDK48 with XbaI (NEBuffer 2 + BSA) and PstI (NeBuffer 3 + BSA) to check wehther the vector is the right one | ||
* digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gel extraction | * digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gel extraction | ||
| - | * Ligation of Fusion-1 fragment and pDk48 plasmid and transformation into | + | * Ligation of Fusion-1 fragment and pDk48 plasmid and transformation into DH5α competent cells |
=== LuxS Test === | === LuxS Test === | ||
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** PCR cycle: 5min @ 98°C || 10s @ 98°C | 30s @ 50°C | 1min @ 72°C || 5min @ 72°C | 4°C hold (30 cycles) | ** PCR cycle: 5min @ 98°C || 10s @ 98°C | 30s @ 50°C | 1min @ 72°C || 5min @ 72°C | 4°C hold (30 cycles) | ||
** NO PCR product | ** NO PCR product | ||
| - | * preparation of | + | * preparation of pTrc99α and pDK48 O/N cultures for Maxipreps |
== Thursday, 08/28/2008 == | == Thursday, 08/28/2008 == | ||
| - | * Maxiprep of pDK48 and | + | * Maxiprep of pDK48 and pTrc99α (product eluted in TB buffer, not water!!) |
* Digestion of LuxQ-Tar fusion (A,B) and LuxQ 1-10 with the follwing enzymes: | * Digestion of LuxQ-Tar fusion (A,B) and LuxQ 1-10 with the follwing enzymes: | ||
*Q(1-10): | *Q(1-10): | ||
Latest revision as of 14:03, 29 October 2008
Contents |
Monday, 08/25/2008
- sequentiell and simultaneous digestion of pDk48 NcoI/NdeI and NcoI/KpnI
--> not successful
- preparation of O/N culture for pDK48 cells
Tuesday, 08/26/2008
Cloning
- test digestion of pDK48 with XbaI (NEBuffer 2 + BSA) and PstI (NeBuffer 3 + BSA) to check wehther the vector is the right one
- digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gel extraction
- Ligation of Fusion-1 fragment and pDk48 plasmid and transformation into DH5α competent cells
LuxS Test
Material: 500 mM IPTG-stock (A), 10 mM IPTG-stock (B)
- negative control: just 1 ml cell suspension
- 10 µM IPTG induced (1 µl stock B + 999 µl cell suspension)
- 20 µM IPTG induced (2 µl stock B + 998 µl cell suspension)
- 50 µM IPTG induced (5 µl stock B + 995 µl cell suspension)
- 100 µM IPTG induced (10 µl stock B + 990 µl cell suspension)
- 500 µM IPTG induced (1 µl stock A + 999 µl cell suspension)
- 1 mM IPTG induced (2 µl stock A + 998 µl celll suspension)
Measurement was done like described in the Materials & Methods section
Wednesday, 08/27/2008
- PCR of LuxQ from V. harveyi with Phusion Mastermix
- PCR cycle: 5min @ 98°C || 10s @ 98°C | 30s @ 50°C | 1min @ 72°C || 5min @ 72°C | 4°C hold (30 cycles)
- NO PCR product
- preparation of pTrc99α and pDK48 O/N cultures for Maxipreps
Thursday, 08/28/2008
- Maxiprep of pDK48 and pTrc99α (product eluted in TB buffer, not water!!)
- Digestion of LuxQ-Tar fusion (A,B) and LuxQ 1-10 with the follwing enzymes:
- Q(1-10):
- XbaI (NEBuffer 2 + BSA) --> if insert: 736 + 1204 bp band; if no insert: 4176 bp band
- EcoRV (NEBuffer 3 + BSA) --> if insert: 5074 bp + 866 band; if no insert: 4176 bp
- F1:
- BamHI (NEBuffer 3 + BSA) --> if insert: 6101 bp band; if no insert: 5768 + 833 bp band
- PstI (NEBuffer 3 + BSA --> if insert: 6101 bp; if no insert: 5768 + 833 bp band
--> No result
