Paris/October 1
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| + | == 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> | ||
| + | <br> | ||
| + | - 72°C final elongation : 7' | ||
Revision as of 20:45, 1 October 2007
Sequencing reactions
In order to verify the quality of the ligation reaction products (of Sep. 18), the following sequencing reactions are being performed
TL_L44.1+O18 TL_L44.1+O19 TL_L44.2+O18 TL_L44.2+O19 TL_L45.1+O18 TL_L45.1+O19 TL_L45.2+O18 TL_L45.2+O19 TL_L46.1+O18 TL_L46.1+O19 TL_L47.1+O18 TL_L47.1+O19 TL_L53.7+O18 TL_L53.7+O19 TL_L53.7+O26 TL_L54.3+O18 TL_L54.3+O19 TL_L54.3+O26 TL_L54.4+O18 TL_L54.4+O19 TL_L54.4+O26 TL_L56.3+O18 TL_L56.3+O19 TL_L56.3+O25 TL_L56.4+O18 TL_L56.4+O19 TL_L56.4+O25 TL_L57.3+O18 TL_L57.3+O19 TL_L57.3+O25 TL_L57.4+O18 TL_L57.4+O19 TL_L57.4+O25 TL_L58.5+O18 TL_L58.5+O19 TL_L58.7+O18 TL_L58.7+O19 TL_L59.6+O18 TL_L59.6+O19 TL_L59.6+O40 TL_L63.1+O18 TL_L63.1+O19 TL_L63.2+O18 TL_L63.2+O19 TL_L64.1+O18 TL_L64.1+O19 TL_L64.1+O25 TL_L64.2+O18 TL_L64.2+O19 TL_L64.2+O25
pDapA DAP-dependant repression through FACS analysis
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"
- 72°C final elongation : 7'
