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

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Contents

Monday, 09/08/2008

  • Liquid Culture of 5 colonies from LuxQ Transformation and 1 from F1 transformation
  • Miniprep of Liquid Culture from previous Transformation (done by Chenchen). Incubated for two days @ RT and about 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 yielded 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
  • 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
  • Gelextraction, eluted in 30 µl
  • Ligation (Insert:Vector 1:1) and transformation into DH5a competent cells

Friday, 09/12/2008

nothing to report


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