Paris/October 1
From 2007.igem.org
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== PCR on Wanner Bfr constructions (L58.5 &L58.7) with the Bfr-Ftsk primers (O43 & O44) == | == PCR on Wanner Bfr constructions (L58.5 &L58.7) with the Bfr-Ftsk primers (O43 & O44) == | ||
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and<br> | and<br> | ||
- 72°C final elongation : 7' | - 72°C final elongation : 7' | ||
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| + | == pDapA DAP-dependant repression through FACS analysis == | ||
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| + | [[Image:pDapA FACS.jpg|thumb|800px|]] | ||
Revision as of 14:43, 3 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
