Team:Paris/September 5


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Purification of yesterday digestion

D112 before gel excision
Digestion n° DNA substrat Digestion by
D112 MP106: S03879 (rbs-TetR) in pSB1A2 EcoRI & SpeI

Electrophoresis and gel extraction

1% agarose gel

Expected size Measured size
pSB1A2 (vector) 2079 bp 2 kb
rbs-TetR (insert) 703 bp 0,7 kb

Qiaquick Gel Extraction Kit

  • elution in 30 µL of EB buffer

Concentration measurement

Sample DNA concentration A260/A280
D112 8 µg/mL 2,31


Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png

List of the constructions used to transformate

Ligation n° Insert Vector Construction
L173 D112 D187 rbs-tetR-mRFP-LVA+
L183 D134 D187 pFlhB - mRFP LVA+
L184 D149 D198 pFliL - ECFP LVA+
L185 D149 D199 pFliL - ECFP LVA-

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