Purification of yesterday digestion

D112 before gel excision

Electrophoresis and gel extraction

1% agarose gel

Qiaquick Gel Extraction Kit

• elution in 30 µL of EB buffer

Concentration measurement

Construction of pFlhB-mRFP-LVA-tripart & pFliL-ECFP-LVA-tripart

• L183 = pFlhB - mRFP LVA+
• L184 = pFliL - ECFP LVA+
• L185 = 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