From 2007.igem.org

yesterday -- tomorrow

PCR on Wanner Bfr constructions (L58.5 &L58.7) with the Bfr-Ftsk primers (O43 & O44)

• We used the Phusion high-fidelity DNA Polymerase (Finnzymes) :
  

- 26,5µL H2O
- 10µL 5X Buffer
- 7µL DNA template
- 2*2,5µL Primers
- 1µL dNTP
- 0,5µL Polymerase

• 2-step PCR reaction : the Tm was higher than 72°C, then Finnzymes asks for us to not use an annealing time anymore.
  

- 98°C initial denaturation : 1'30
30 steps of :
- 98°C denaturation : 30"
- 72°C elongation : 50"
and
- 72°C final elongation : 7'

pDapA DAP-dependant repression through FACS analysis

Strain FR781 containing a plasmid with the mRFP expression controlled by the promotor pDapA has been cultured overnight in 5 mL of LB with different concentrations of DAP.

- 1mM
- 300 µM
- 100 µM
- 50 µM

- 0 µM environment has been simulated by dilluting and letting overnight 300µM DAP LB culture at 1/100 in minimal medium M9 without DAP during 1h before analysis at 4°C.

All analyses are made on 1/100 dillution of overnight LB culture in minimal medium M9 at 4°C.

Remark : values from the 50µM DAP culture has not been put on the graph since it didn't give linear results regarding other concentrations (Mean Fluo = 9,41). I don't what to think, it is possible that taking into consideration the fact that 50µM is a limitant concentration for growth (bacteria divided slower than the others) it would affect the pDapA repression.