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Raphaël

Gel extraction

• Left arm HK022 Mut-Mut
• Left arm HK022 WT-Mut
• Left arm λ Mut-Mut
• Left arm λ WT-Mut
• Left arm WT-WT
• Right arm HK022 Mut-Mut
• Right arm HK022 WT-Mut
• Right arm λ Mut-Mut
• Right arm λ WT-Mut

PCR purification

• Kan resistance cassette
• LacZα expression cassette

Ligations

• 1)Left arm HK022 Mut-Mut + Kan resistance cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
• 2)Left arm HK022 WT-Mut + Kan resistance cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
• 3)Left arm λ Mut-Mut + Kan resistance cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
• 4)Left arm λ WT-Mut + Kan resistance cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
• 5)Left arm WT-WT + Kan resistance cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
• 6)Right arm HK022 Mut-Mut + LacZα expression cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
• 7)Right arm HK022 WT-Mut + LacZα expression cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
• 8)Right arm λ Mut-Mut + LacZα expression cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
• 9)Right arm λ WT-Mut + LacZα expression cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)

Controls:

• 13)Kan resistance cassette (1µl + 0.5µl ligase + 1µl buffer + 9µl water)
• 14)LacZα expression cassette (1µl + 0.5µl ligase + 1µl buffer + 9µl water)

Transformations of DH5α with 2µl of miniprep, plating with 150µl

• Left arm HK022 Mut-Mut
• Left arm HK022 WT-Mut
• Left arm λ Mut-Mut
• Left arm λ WT-Mut
• Left arm WT-WT
• Right arm HK022 Mut-Mut
• Right arm HK022 WT-Mut
• Right arm λ Mut-Mut
• Right arm λ WT-Mut
• 17)pUC19 (positive control)

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Raphaël

Restriction digest with SpeI and PstI : 10µl miniprep + 10µL water + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA

• Ps/wRBS/LuxR + attC/term/attC/mRFP
• Ps/mRBS/LuxR + attC/term/attC/mRFP

Restriction digest with XbaI and PstI : 10µl miniprep + 10µL water + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA

• wRBS/TetA(C)
• mRBS/TetA(C)
• RBS/LuxI