"
Team:Heidelberg/Notebook/Sensing Group/Notebook/4thweek
From 2008.igem.org
(Difference between revisions)
(→Thursday, 08/28/2008) |
(→Wednesday, 08/27/2008) |
||
| Line 151: | Line 151: | ||
== Wednesday, 08/27/2008 == | == Wednesday, 08/27/2008 == | ||
* PCR of LuxQ from ''V. harveyi'' with Phusion Mastermix | * 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 | + | ** 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 | --> NO PCR product | ||
* preparation of pTrc99a and pDK48 O/N cultures for Maxipreps | * preparation of pTrc99a and pDK48 O/N cultures for Maxipreps | ||
Revision as of 20:09, 26 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 of pDK48 transformed cells
Tuesday, 08/26/2008
Cloning
- 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
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: 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
Thursday, 08/28/2008
- 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):
- XbaI --> if insert: 736 + 1204 bp band; if no insert: 4176 bp band
- EcoRV --> if insert: 5074 bp + 866 band; if no insert: 4176 bp
- F1:
- BamHI --> if insert: 6101 bp band; if no insert: 5768 + 833 bp band
- PstI --> if insert: 6101 bp; if no insert: 5768 + 833 bp band
--> No result
