Tuesday 30^th august

Visualization

Plasmids extraction

"By Mahnaz"

Plasmid pZA11 were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit

Gibson Assembly of segments 1 and 2

By Mahnaz

Two preparations were made :

• the 1-2 Gibson assembly product tube : 1,12µL of purified segment 1 and 0,56µL of purified segment 2 + 0,85µL of digested pSB1C3 + 7,47 µL of sterile water + 10µL of NEB Builder Hifi DNA Assembler Master Mix
• the control tube : 0,85µL of digested pSB1C3 + 17,47 µL of sterile water

Gibson products were incubated 1h at 52°c.

Transformation in DH5α with the 1-2 Gibson assembly product

By Mahnaz

The transformation was performed following the usual protocol with 2µL of plasmid.

• 50, 150 and 350µL of the transformed DH5α with the 1-2 Gibson assembly product were plated on 3 plates of LB + chloramphenicol (30µg/mL)
• 350 µL of each Gibson assembly control and transformation control were plated on 2 plates of B + chloramphenicol (30µg/mL)

Petri cultures were incubated at 37°c overnight.