Team:Heidelberg/Notebook/Sensing Group/Notebook/1stweek

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(Difference between revisions)
(Cloning of LuxP)
(Monday, 08/04/2008)
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=== Cloning of LuxP ===
=== Cloning of LuxP ===
[[Image:HD_080805-LuxP_PCR.png|right|thumb|150px|LuxP colony PCR]]
[[Image:HD_080805-LuxP_PCR.png|right|thumb|150px|LuxP colony PCR]]
 +
[[Image:HD 080805-LuxQ S PCR.png|right|thumb|150px|LuxQ, LuxS PCR]]
* PCR of LuxP from ''V. harveyi'' colony
* PCR of LuxP from ''V. harveyi'' colony
* Gel Purification of LuxP eluted in 30 µl H<sub>2</sub>O
* Gel Purification of LuxP eluted in 30 µl H<sub>2</sub>O
* Digestion of LuxP with SalI and NotI of pDK48 and LuxP-Gel-Purification product: 2 µl NEB-Buffer 3 + 0.5 µl SalI/NotI + 10 µl DNA + 7 µl H<sub>2</sub>O
* Digestion of LuxP with SalI and NotI of pDK48 and LuxP-Gel-Purification product: 2 µl NEB-Buffer 3 + 0.5 µl SalI/NotI + 10 µl DNA + 7 µl H<sub>2</sub>O
 +
* Ligation with vector:insert ratio 1:3 and 1:1
 +
 +
=== Cloning of LuxS ===
 +
* PCR of ''V. harveyi'' genome and PCR purification
 +
* digestion of LuxS and pTrc99a with BamHI/NcoI
 +
* Ligation with vector:insert ratio 1:3 and 1:1
== Tuesday, 08/05/2008 ==
== Tuesday, 08/05/2008 ==

Revision as of 09:59, 26 October 2008

Back to the overview

Contents

Monday, 08/04/2008

Preparations

Preparations were done at the end of the week before

  • Transformation of pDK48 and pTrc99a in E. coli DH5a (0.5 µl plasmid-DNA + 50 µl competent cells)
  • Glycerol-stock of Vibrio harveyi BB120, BB886, mm30, BB178, BB125
  • picked pTrc99a and pDK48 cultures and inoculated in 3 ml LB overnight
  • V. harveyi cultured on LB+-Agar plate
  • MiniPrep of pTrc99a and pDK48 from DHha

Cloning of LuxP

LuxP colony PCR
LuxQ, LuxS PCR
  • PCR of LuxP from V. harveyi colony
  • Gel Purification of LuxP eluted in 30 µl H2O
  • Digestion of LuxP with SalI and NotI of pDK48 and LuxP-Gel-Purification product: 2 µl NEB-Buffer 3 + 0.5 µl SalI/NotI + 10 µl DNA + 7 µl H2O
  • Ligation with vector:insert ratio 1:3 and 1:1

Cloning of LuxS

  • PCR of V. harveyi genome and PCR purification
  • digestion of LuxS and pTrc99a with BamHI/NcoI
  • Ligation with vector:insert ratio 1:3 and 1:1

Tuesday, 08/05/2008

Wednesday, 08/06/2008

Thursday, 08/07/2008

Friday, 08/08/2008