Notebook : July 2

summary

• Made a quantity control for fnr/PSB1C3 ligation products by using NanoDrop

• performed 4 genetic transformations: LacZ(with plasmid PSB1A2) , AmilCP(with plasmid PSB1C3) and the products of ligation (fnr+plasmid PSB1C3) into competent cells. The 4th transformation was a control sample.

• cloning. Plated the products of these 4 transformations strains in liquor and on solid environment with 2 antibiotics: ampicillin and chloramphenicol (Amp->LacZ; CM->AmilCP).

Lab work

• A.aero/anaerobic regulation system
  
  
  • 1.BioBrick promotor fnr(repressor) in plasmid PSB1C3

Ligation product quantification

We used the nanodrop to measure the DNA in 260nm and we found its concentration is 411.7ng/µl.

• A.aero/anaerobic regulation system
  • 2.BioBrick RBS+LacZ+terminator in plasmid PSB1C3
  • BioBrick RBS+amilCP+terminator in plasmid PSB1C3

Transformation

We performed 4 transformations: LacZ with plasmid PSB1A2( RBST 3K plate 2 2012 suspended in 10µl H2O) , AmilCP(with plasmid PSB1C3), the products of ligation (fnr+plasmid PSB1C3) and a control.

And we added in each sample 50µl competent cells.

After thermal shock, those sample were mixed with 1ml LB medium, incubated at 37 °C during 1h30.

Cloning

We prepared Petri dishes by 2 different mediums:

• 500ml LB+0.5ml chloramphenicol for AmilCP, mixture(fnr) and control
• 250ml LB+0.25ml ampicillin for LacZ

Then the products of transformations were seed on the medium:

• 1.Seed directly 100µL of each one on their medium
• 2.Seed indirectly after concentration of products of transformation(centrifuge->remove supernatant->resuspend)

The incubation lasted one night.