Construction of rbs-TetR-mRFP-LVA-tripart (L173)

Transformation

Ligation L173:

• insert: D112 (ES) rbs-TetR
• vector: D187 (EX) mRFP-LVA-tripart

Insert:vector ratio : 3:1 or 4:1

Transformation of E. coli DH5 alpha competent cells

• Defroze of the cells on ice
• Add 5 µL of DNA in 100 µL of competent cells (ligation products or pUC19 for positive control)
• 30 min on ice
• Heat schock 45sec at 42°C
• 2 min on ice
• Add 900 µL of SOC
• Incubate 1h at 37°C
• Centrifugate 5 min at 6000 rpm
• Remove 800 µL
• Plate the 200 µL left on LB + amplicilline
• incubate O/N at 37°C