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

Monday, 08/25/2008

--> not successful

Digestion of pDK48 with xbaI and PstI yield expected results

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

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 pTrc99α and pDK48 O/N cultures for Maxipreps

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):
1. XbaI (NEBuffer 2 + BSA) --> if insert: 736 + 1204 bp band; if no insert: 4176 bp band
2. EcoRV (NEBuffer 3 + BSA) --> if insert: 5074 bp + 866 band; if no insert: 4176 bp
F1:
1. BamHI (NEBuffer 3 + BSA) --> if insert: 6101 bp band; if no insert: 5768 + 833 bp band
2. PstI (NEBuffer 3 + BSA --> if insert: 6101 bp; if no insert: 5768 + 833 bp band

--> No result