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

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(Cloning)
(Thursday, 08/28/2008)
 
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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 pTrc99a and pDK48 O/N cultures for Maxipreps
+
* preparation of pTrc99α and pDK48 O/N cultures for Maxipreps
== Thursday, 08/28/2008 ==
== Thursday, 08/28/2008 ==
-
* Maxiprep of pDK48 and pTrc99alpha (product eluted in TB buffer, not water!!)
+
* 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

Back to the overview

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

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

LuxS Test

Material: 500 mM IPTG-stock (A), 10 mM IPTG-stock (B)

  1. negative control: just 1 ml cell suspension
  2. 10 µM IPTG induced (1 µl stock B + 999 µl cell suspension)
  3. 20 µM IPTG induced (2 µl stock B + 998 µl cell suspension)
  4. 50 µM IPTG induced (5 µl stock B + 995 µl cell suspension)
  5. 100 µM IPTG induced (10 µl stock B + 990 µl cell suspension)
  6. 500 µM IPTG induced (1 µl stock A + 999 µl cell suspension)
  7. 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):
    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

Friday, 08/29/2008

Go to 5th week