Paris/October 1
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| - | == | + | [[Paris/September 30|yesterday]] -- [[Paris/October 2|tomorrow]] <br> |
| + | == 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) :<br> | |
| - | + | <br> | |
| - | + | - 26,5µL H2O<br> | |
| - | + | - 10µL 5X Buffer<br> | |
| - | + | - 7µL DNA template<br> | |
| - | + | - 2*2,5µL Primers<br> | |
| - | + | - 1µL dNTP<br> | |
| - | + | - 0,5µL Polymerase<br> | |
| - | + | <br> | |
| - | + | * 2-step PCR reaction : the Tm was higher than 72°C, then Finnzymes asks for us to not use an annealing time anymore.<br> | |
| - | + | <br> | |
| - | + | - 98°C initial denaturation : 1'30<br> | |
| - | + | 30 steps of : <br> | |
| - | + | - 98°C denaturation : 30"<br> | |
| - | + | - 72°C elongation : 50"<br> | |
| - | + | and<br> | |
| - | + | - 72°C final elongation : 7' | |
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== pDapA DAP-dependant repression through FACS analysis == | == 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.<br> | ||
| + | <br> | ||
| + | - 1mM<br> | ||
| + | - 300 µM<br> | ||
| + | - 100 µM<br> | ||
| + | - 50 µM<br> | ||
| + | <br> | ||
| + | - 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.<br> | ||
| + | <br> | ||
| + | All analyses are made on 1/100 dillution of overnight LB culture in minimal medium M9 at 4°C.<br> | ||
| + | |||
| + | 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.<br> | ||
| + | |||
| + | [[Image:pDapA FACS.jpg|thumb|800px|]] | ||
Latest revision as of 18:08, 7 October 2007
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.
