Team:Paris Saclay/Notebook/July/4

From 2013.igem.org

(Difference between revisions)
(→1 - Estimation of Pfnr Colony PCR size fragments)
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===='''Objective : obtaining Bba_K1155000'''====
===='''Objective : obtaining Bba_K1155000'''====
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===='''1 - Estimation of Pfnr Colony PCR size fragments'''====
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===='''1 - Estimation of Pndh* Colony PCR size fragments'''====
Abdou, Anaïs, Sheng
Abdou, Anaïs, Sheng
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Estimated size :  
Estimated size :  
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* VF/VR primer -> Plasmid without Pfnr size : 277bp
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* VF/VR primer -> Plasmid without Pndh* size : 277bp
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* VF/Pfnr_down -> Plasmid with Pfnr size : 276bp
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* VF/Pfnr_down -> Plasmid with Pndh* size : 276bp
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* Pfnr_Up/VR -> Plasmid with Pfnr size : 311bp
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* Pfnr_Up/VR -> Plasmid with Pndh* size : 311bp
===='''2 - Electrophoresis of Colony PCR products'''====
===='''2 - Electrophoresis of Colony PCR products'''====

Revision as of 13:49, 29 September 2013

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Contents

Notebook : July 4

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining Bba_K1155000

1 - Estimation of Pndh* Colony PCR size fragments

Abdou, Anaïs, Sheng

We used software gene manager to find the correct size of our fragments.

Estimated size :

  • VF/VR primer -> Plasmid without Pndh* size : 277bp
  • VF/Pfnr_down -> Plasmid with Pndh* size : 276bp
  • Pfnr_Up/VR -> Plasmid with Pndh* size : 311bp

2 - Electrophoresis of Colony PCR products

Zhou

Psgel0407.jpg
  • Well 1 to 4 : 5µL of PCR products with VF/VR primers+1µL of 6X loading dye
  • Well 5 to 6 : 5µL of PCR product withVF/Pfnr_Down primers+1µL of 6X loading dye
  • Well 7 : 6µL of DNA Ladder
  • Well 8 to 9 : 5µL of PCR product withVF/Pfnr_Down primers+1µL of 6X loading dye
  • Well 10 to 13 : 5µL of PCR products with Pfnr_Up/VR primers+1µL of 6X loading dye
  • Well 14 : 6µL of DNA Ladder
  • Gel : 1.5%

We obtain fragment at the right size in wells : 3, 4 , 8 , 9 12 and 13. We will digest our biobrick to confirm that Pfnr insert PSB1C3 correctly.

3 - Stock of Bba_K1155000

Zhou

Used quantities :

  • Bba_K1155000 confirmed : 1 mL
  • Glycerol : 500µL glycerol.

We stocked them at -20°C.

4 - Extraction of Bba_K1155000 from DH5α

Anaïs, Sheng

Protocol : Hight copy plamid extraction

5 - Digestion of Bba_K1155000 by NotI, MluI, HpaI to check good insertion of Pfnr in PSB1C3

Abdou

Used quantities :

  • Bba_K1155000 : 2µL
  • Bufer oranger : 2µL
  • NotI or MluI or HpaI : 0.5µL
  • H2O : 15.5µL

We let the digestion 1h30 at 37°C.

6 - Culture of Bba_K1155000

Anaïs, Sheng

We made 2 cultures of bacterias transformed with Bba_K1155000 that show a fragments at the right size at electrophoresis.


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