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

Revision as of 20:10, 26 October 2008 by Koljaschleich (Talk | contribs)

Monday, 08/25/2008

--> not successful

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
digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gelextraction
Ligation of Fusion-1 and pDk48 and transformation into DH5a 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: 98 °C, 5 min |98 °C, 10 sec --- 50 °C, 30 sec --- 72 °C, 1 min|30x --- 72 °C, 5 min --- 4 °C, forever
- 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