Team:Paris/September 4

=Construction of rbs-TetR-mRFP-LVA-tripart (L173)=

Transformation results (rbs-TetR in mRFP-LVA-tripart-pSB1A3)
Ligation L173
 * insert: D112 (ES) rbs-TetR
 * vector: D187 (EX) mRFP-LVA-tripart-pSB1A3

PCR Screening


PCR screening programm
 * elongation tim: 2 min
 * primers used: O18 & O19
 * positive PCR control: S158 (pSB3K3)
 * negative PCR control: no template

Results: the clones are not correct.

=Digestion=

Incubation 2h30 at 37°C and then 20 min at 65°C. Not purified yet. Put at -20°C.
 * 5 µL of DNA
 * 3 µL of 10X buffer n°2
 * 0,3 µL 100X BSA
 * 1 µL of EcoRI
 * 1 µL of SpeI
 * 19,7 µL of water

=Construction of pFlhB-mRFP-LVA-tripart & pFliL-ECFP-LVA-tripart=


 * L183 = pFlhB - mRFP LVA+
 * L184 = pFliL - ECFP LVA+
 * L185 = pFliL - ECFP LVA-

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

=>the measurement of the absorbance was so instable, that we have prefered to do it again, we do it by an another thechnik : Analysis on gel.

Ligation
Protocol

=>it has been missed to do adaptated control.