Team:Paris/August 22

=Analysis of the transformant of FlhDC+promotor=


 * Analysis of the plasmids MP160.1 and MP160.2 (FlhDC+promotor in pSB1A2)
 * Control: MP143 (GFP generator in pSB1A2)

PCR
PCR screening programm elongation time: 1 min 30 total volume reaction (25 µL)
 * 12,5 µL Quick load PCR Mix 2X
 * 0,5 µL O18
 * 0,5 µL O19
 * 1 µL DNA
 * 10,5 µL water

Digestion
total volume reaction (30 µL) Incubation 2h55 at 37°C and then 20 min at 65°C.
 * 2 µL DNA
 * 3 µL buffer 2 10X
 * 0,3 µL BSA 100X
 * 1 µL XbaI
 * 1 µL SpeI
 * 22,7 µL water

Electrophoresis



 * 1% agarose gel
 * for PCR products: 10 µL loaded
 * for digestion products (30 µL): adding of 7 µL of loading blue and then 20 µL loaded on gel

Results: The clones tested didn't have the insert.

=Construction of pFlgA - GFP Generator=

Aim : Construction of  "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0240)

Digestion
Protocol Digestion

Gel Extraction
Protocol

Measurement of the concentration of D168 purified
Protocol (it's same that for Miniprep)

Ligation
Protocol

=Construction for FIFO=

Aim : Construction of pFlgA - YFP tripart (+/- LVA) "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)

Transformation of the ligations we did yesterday
Protocol

=Construction for synchronization=

Transformation of the ligations we did yesterday
Protocol

Ligation
Protocol

Transformation
Protocol

=Promoter characterization plasmids=

Transformation of ligations from August 20th
Protocol

Top 10 cells were used