Team:Paris/September 3

From 2008.igem.org

← Yesterday

↓ Calendar ↑

Tomorrow →

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

Part icon rbs.pngIcon coding.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png

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