Team:Heidelberg/Notebook/Sensing Group/Notebook/4thweek

(Difference between revisions)

Revision as of 22:59, 26 October 2008

--> not successful

Digestion of pDK48 with xbaI and PstI yield expected results

test digestion of pDK48 with XbaI and PstI 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 DH5alpha competent cells

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

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 pTrc99a and pDK48 O/N cultures for Maxipreps

Maxiprep of pDK48 and pTrc99alpha (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):
1. XbaI --> if insert: 736 + 1204 bp band; if no insert: 4176 bp band
2. EcoRV --> if insert: 5074 bp + 866 band; if no insert: 4176 bp
F1:
1. BamHI --> if insert: 6101 bp band; if no insert: 5768 + 833 bp band
2. PstI --> if insert: 6101 bp; if no insert: 5768 + 833 bp band

--> No result

@@ Line 132: / Line 132: @@
 === Cloning ===
 [[Image:HD 080826-pDK48 digestions.png|right|thumb|200px|Digestion of pDK48 with xbaI and PstI yield expected results]]
-* digestion of pDK48 with XbaI and PstI to check wether the vector is the right one
+* test digestion of pDK48 with XbaI and PstI to check wehther the vector is the right one
-* digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gelextraction
+* digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gel extraction
-* Ligation of Fusion-1 and pDk48 and transformation into DH5a competent cells
+* Ligation of Fusion-1 fragment and pDk48 plasmid and transformation into DH5alpha competent cells
 === LuxS Test ===