Team:Heidelberg/Notebook/Sensing Group/Notebook/2ndweek

From 2008.igem.org

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[[Image:HD 080815-F1 digestion pDK48.png|left|thumb|250px|Gelextraction of digested Fusion-1 and purified PCR product as well as pDK48 digested. Expected bands at 516bp and 392bp are not visible.]]
[[Image:HD 080815-F1 digestion pDK48.png|left|thumb|250px|Gelextraction of digested Fusion-1 and purified PCR product as well as pDK48 digested. Expected bands at 516bp and 392bp are not visible.]]
[[Image:HD 080815-F1 digestion NcoI NdeI.png|left|thumb|200px|Fusion-1 digested with NcoI/NdeI. Expected bands of 1025bp and 908bp had to be cut out together. Wanted band is the longer one.]]
[[Image:HD 080815-F1 digestion NcoI NdeI.png|left|thumb|200px|Fusion-1 digested with NcoI/NdeI. Expected bands of 1025bp and 908bp had to be cut out together. Wanted band is the longer one.]]
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* Ligation of 5 µl Fusion-1, digested with NcoI/NdeI and 5 µl pDK48
* Ligation of 5 µl Fusion-1, digested with NcoI/NdeI and 5 µl pDK48
* Transformation into DH5a (5 µl Ligation & 15 µl Ligation)
* Transformation into DH5a (5 µl Ligation & 15 µl Ligation)
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[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/3rdweek | Go to 3rd week]]
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/3rdweek | Go to 3rd week]]

Revision as of 13:43, 26 October 2008

Back to the overview

Contents

Monday, 08/11/2008

  • preparation of LuxS harboring cells O/N culture

Tuesday, 08/12/2008

  • Miniprep of correctly sequenced LuxS and Glycerol-stock
  • LuxQ no. 3 checked via digestion with NcoI/BamHI --> negative result
  • PCR for LuxQ, LuxQ-1, LuxQ-2, Tar-1, Tar-2 with Taq Mastermix
  • preparation of O/N culture for LuxQ no. 3

Wednesday, 08/13/2008

  • PCR-purification of the 5 PCR-products eluted in 50 µl H2O
  • Miniprep of LuxQ no. 3 O/N culture and digestion with xbaI
  • Fusion-PCR for Fusion-1 and repetetion of PCR for LuxQ-2 and LuxQ
  • digestion of pDK48 and Fusion-1 with NcoI/NdeI (0.5 µl) and AgeI (1 µl) in NEBuffer 4
Fusion-PCR for Fusion-1, LuxQ PCR, LuxQ-2 PCR, pDK48 miniprep and xbaI digestion of LuxQ no. 3
Digestion of Fusion-1 and pDK48 with NcoI/NdeI/AgeI


Thursday, 08/14/2008

PCR for LuxQ and LuxQ-2
  • Gel from LuxQ, LuxQ2
  • sequentiell digestion of Fusion-1 with NcoI and NdeI (NEBuffer 3)
  • Double digestion of pDK48 with NcoI/NdeI (NEBuffer 4)


Friday, 08/15/2008

  • digestion of Fusion-1 with AgeI in NEBuffer 1
  • Gelextraction of sequentially digested Fusion-1 and pDK48
  • new digestion of Fusion-1 with NcoI/NdeI and Gelextraction, because previous digestion did not yield expected bands (there should be two bands at 516bp and 392bp)
Gelextraction of digested Fusion-1 and purified PCR product as well as pDK48 digested. Expected bands at 516bp and 392bp are not visible.
Fusion-1 digested with NcoI/NdeI. Expected bands of 1025bp and 908bp had to be cut out together. Wanted band is the longer one.


  • Ligation of 5 µl Fusion-1, digested with NcoI/NdeI and 5 µl pDK48
  • Transformation into DH5a (5 µl Ligation & 15 µl Ligation)


Go to 3rd week

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