Team:Paris/August 25

=Construction for Synchronization=

Transformation of the ligations we did yesterday
=Construction of pFlgA - GFP Generator=

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

Results of the transformation we did the day before yesterday
=> Need to screen to know which clones we can use for the of  pFlgA promotor characterization.

=Cloning of EnvZ*= The sequencing of EnvZ* previously cloned, revealed a loss of about 300 bp. EnvZ* contains indeed an EcoRI restriction site within its sequence. So we can't use this enzyme during the cloning.

Digestion
Reaction mixture Incubation at 37°C during 2H25, and then ~20 min at 65°C
 * 4 µL of PCR129 (or 2 µL of MP108)
 * 3 µL of 10X buffer n°2
 * 0,3 µL of 100X BSA
 * 1 µL of XbaI
 * 1 µL of PstI
 * 20,7 µL (or 22,7 µL) of water

Electrophoresis


1% agarose gel
 * EnvZ*: 3 µL of digestion products + 1 µL of loading blue + 2 µL of water
 * pSB1A2: 30 µL of digestion products + 6 µL of loading blue

Purification elution in 30 µL of buffer EB then kept at - 20°C
 * EnvZ*: digestion product purified directly by Qiagen kit
 * pSB1A2: excision of the band (2 kb) from the gel and purification by the Qiaquick Gel Extraction kit

=Screening of the cloning of pFlgA-YFP Tripart (LVA+/-)=

Minipreps and glycerol stock
=Promoter characterization plasmids=

PCR screenning: transformation results from August 23th
Protocol