Team:Paris/August 1

souche stockage

MiniPreps

Conditions

• Use of Quiagen's protocol on all the clones cultivated on the 31th.
• Preparation of 50µl of minipreps in water.

Stock Glycerol =

Protocol

• 1mL glycerol
• 1mL LB

S123	L104	Strong rbs - lasR activator D129 (BV) - D114 (BI)
S124	L105	Strong promoter - ECFP D123 (BV) - D130 (BI)
S125	L106	Strong promoter - gfp Tripart D123 (BV) - D131 (BI)
S126	L107	Strongest promoter - ECFP D103 (BV) - D131 (BI)
S127	L108	Strong promoter - gfp Tripart D103 (BV) - D130 (BI)
S128	L110	Medium promoter - gfp Tripart D124 (BV) - D131 (BI)
S129	L111	Weak promoter - ECFP D104 (BV) - D130 (BI)
S130	L112	Weak promoter - gfp D104 (BV) - D131 (BI)
S131	L115	pLas - ECFP D105 (BV) - D130 (BI)
S132	L116	pLas - gfp Tripart D105 (BV) - D131 (BI)
S133	L117	Double Terminator - yfp D125 (FV) - D117 (FI)
S134	L118	Double Terminator - rfp D125 (FV) - D121 (FI)
S135	L119	Double Terminator - lasR activator + LVA D125 (FV) - D113 (FI)
S136	L121	Strong promoter - gfp tripart D106 (BV) - D130 (BI)
S137	L124	Strongest RBS - rfp D102 (BV) - D122 (BI)
S138	L125	Strongest RBS - yfp D102 (BV) - D118 (BI)

Transformations

Protocol

use of TOP10 Chemically competent cells

• Defroze competent cells on ice during 5'
• Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
• Incubate 30' on ice
• Heat-shock the cells during 30" at 42°C without shaking
• Put 2' on ice
• Add 250µL of pre-warmed SOC medium (4°C)
• Incubate 1h at 37°C under shaking (225rpm)
• Spin at 5.000rpm during 30"
• Remove 150µL of supernatant
• Resuspent the pellet in the 150µL left
• Spread on adequated plates
• Incubate O/N at 37°C

List of transformation