Team:Paris Saclay/Notebook/July/4

From 2013.igem.org

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 Pndh* 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 Pndh* 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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