Notebook : July 4

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining Bba_K1155000

1 - Estimation of Pfnr 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 Pfnr size : 277bp
• VF/Pfnr_down -> Plasmid with Pfnr size : 276bp
• Pfnr_Up/VR -> Plasmid with Pfnr size : 311bp

2 - Electrophoresis of Colony PCR products

Zhou

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%

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

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.