Team:Heidelberg/Notebook/Sensing Group/Notebook/6thweek

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Contents

Monday, 09/08/2008

  • Culture of 5 colonies from LuxQ Transformation and 1 from F1 transformation
  • Miniprep of Culture from previous Transformation (done by Chenchen). Incubated for two days @ RT and about then 6 h @ 37 °C
  • Digestion of the Miniprep products with XbaI (NEBuffer 2 + BSA, 1 ul enzyme @ 37 °C about 40min)
Digestion of LuxQ with xbaI yielded expected bands (4736 + 1204 bp) for all samples, except for no. 4
Sequencing @ GATC verified correct sequence for LuxQ no. 1
  • PCR for LuxQ and LuxP with Phusion (25 µl Mastermix, 0.5 µl each Primer, 0(1) µl DMSO, 24(23) µl H20, 0.5 µl Template)
  • PCR Programme: 5 min @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C | 30 s @ 72 °C || 5 min @ 72 °C | 4 °C
PCR for LuxQ and LuxP. LuxQ PCR did not work, but first PCR for LuxP was positive.

Tuesdsay, 09/09/2008

  • Miniprep of LuxQ colonies no. 1,2,3,5,6
HD 080909-PCR Miniprep-LuxQ 2.png
HD 080909-PCR Miniprep-LuxQ 1.png


  • PCR for LuxQ fragment 1 and 2, Tar fragment 1 and 2 for Phusion receptor with Phusion-Polymerase
  • PCR Programme: 30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)
PCR of LuxQ and Tar constructs for fusion receptor. LuxQ is negative
PCR of LuxQ constructs for fusion


  • Fusion-PCR for Fusion-1 and Fusion-2 constructs
Fusion-PCR
  • O/N Culture of 10 LuxP in pDK48 colonies

Wednesday, 09/10/2008

LuxP cloning

  • Miniprep of O/N LuxP cultures and digestion with NcoI (NEBuffer 2, 2h @ 37 °C)
LuxP digestion with NcoI. All colonies are positive
Sequencing @ GATC: LuxP1 correct

Fusion chimeras

  • PCR for LuxQ 1+2, Tar 1+2 and Gel Extraction to get rid of first primers
  • Fusion-PCR of LuxQ1+Tar1 und LuxQ2+Tar2 with Phusion and 55 °C annealing --> No product
  • Fusion PCR of LuxQ fragment 1b, LuxQ fragment 1c, LuxQ fragment 2b, LuxQ fragment 2c, Tar fragment 1b, Tar fragment 1c, Tar fragment 2b, Tar fragment 2c with Phusion and 55 °C annealing. Products were purified via Gelextraction to get rid of first primers
  • Two Fusion PCR (30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)) with PCR purification.
  • Gel Extraction of LuxQ 1+2, Tar 1+2
First Fusion PCR
another fusion PCR


Thursday, 09/11/2008

Fusion chimeras

  • PCR Purification of previous Fusion PCR
  • Digestion with NcoI/NdeI (NEBuffer4) and NcoI/KpnI (NEBuffer1 + BSA). 1 h @ 37 °C
  • Gel extraction, eluted in 30 µl water
  • Ligation (Insert:Vector 5 µl:5 µl) and transformation into DH5alpha competent cells

Friday, 09/12/2008

nothing to report


Go to 7th week