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Raphaël

Restriction digest , 2h at 37°C

We want to amplify this DNA, so the idea is to cut the plasmid once to obtain a linear fragment that can be easily amplified with our primers.

• mRBS/TetA(C)+RBS/LuxI: 5µl miniprep + 1µl B4 + 0.1µl BSA + 3µl water + 1µl AlwNI
• wRBS/TetA(C)+RBS/LuxI: 5µl miniprep + 1µl B4 + 0.1µl BSA + 3µl water + 1µl AlwNI
• stRBS/TetA(C)+RBS/LuxI: 5µl miniprep + 1µl B4 + 0.1µl BSA + 3µl water + 1µl AlwNI

PCR
Primers : VF2 + VR

• mRBS/TetA(C)+RBS/LuxI
• wRBS/TetA(C)+RBS/LuxI
• sRBS/TetA(C)+RBS/LuxI
• mRBS/TetA(C)+RBS/LuxI cut by AlwNI
• wRBS/TetA(C)+RBS/LuxI cut by AlwNI
• sRBS/TetA(C)+RBS/LuxI cut by AlwNI

-> PCR tubes stored overnight at -20°C.

Ligation for 1h30 at 25°C

• wLac+LuxI : 1µl 1/2 dilution + 4µl + 1µL buffer + 4µL H20 + 0.5µl ligase
• sLac+LuxI : 1µl 1/4 dilution + 4µl + 1µL buffer + 4µL H20 + 0.5µl ligase

Control with only LuxI has already be done yesterday.

Transformation in Turbocells

With today's ligations:

• wLac+LuxI
• sLac+LuxI

Control :

• positive control (pUC19)

Overnight cultures
From glycerols :

• wRBS/LacZα
• sRBS/LacZα
• RBS/LuxI
• pLux
• attC-t-attC-mRFP
• PmM-LuxR
• PmW-LuxR
• wTet-LuxI
• mTet-LuxI
• sTet-LuxI

From Aleksandra's transformations :

• 1x PmW-LuxR-attC
• 3x pLux-LuxI
• 4x PsS-attC + pBad-Int
• 4x PsM-attC + pBad-Int
• 4x PsW-attC + pBad-Int
• 4x PmS-attC + pBad-Int