Notebook : August 8

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining the PSB3K3 backbone plasmid

1 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion was right

Damir, Nadia

Expected sizes :

• PSB3K3 : 2750kb
• GFP : 1069kb

We obtained fragments of the right size. Now we can purify the PSB3K3 plasmid.

2 - Gel purification of the PSB3K3 plasmid from BBa_J004450 digested by EcoRI/Pst1

2.1 - Migration of the remaining 45µL of BBa_J004450 digested by EcoRI/Pst1

Anaïs

Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.

2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel

Nadia

Protocol : Electroelution

We let the plasmid precipitate during the night.

Obtaining RBS_LacZ+Term_PSB1C3

1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies

Anaïs

• Colony counting :
  • Low concentration petri dish : 47 colonies
  • High concentration petri dish : 145 colonies

• Picking of 25 colonies

• Preparation of 700µL of Master mix
  • H₂O : 590µL
  • dNTP : 28µL
  • VF2 primer : 3.5µL
  • VR primer : 3.5µL
  • DreamTaq buffer 10x : 70µL
  • DreamTaq enzyme : 5µL

Protocol : Colony PCR

PCR Program :
 [IMAGE]

2 - Gel electrophoresis of the colony PCR products

Anaïs, Damir

Expected size : 3583bp

Colonies 10, 14, 15 exhibit plasmids with the right length.