From 2007.igem.org

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"
and
- 72°C final elongation : 7'