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From 2014.igem.org

10^th July

• SLP gene synthesis
  
  Step 2:
  We made 35 cycles for the PCR instead of 30.
  We made again the calcul of the annealing temperature with the estimation of the concentration in fragment after the first step.
  We extended the time let for the hybridation.
  
  PCR parameter:
  92°C 1min
  35 cycles : 92°C 10s, Gradient 64°C delta 6°C : (2AB) (3) 62°C 1min ; (2CD) (11) 66°C 1min, 72°C 10s
  End at 4°C
  
  
  
  PCR Parameter :
  98°C 5 min
  35 cycles : 98°C 10s, Gradient 61°C delta 18°C : (2AB) (11) 67°C 20s ; (2CD) (12) 68°C 20s (instead of 69°C), 72°C 1min
  Final extension 72°C 10min
  End at 4°C
  
  We obtain this gel
  
  
  
  Step 3: Vtot = 100µL and then 2x50µL
  Estimation of the quantity with the gel to calculate the annealing temperature (Tm)
  20µL phusion buffer 5X
  0,5µL dNTP 10mM
  1µL phusion polymérase
  3,5µL DMSO
  10µL 2AB8°°
  10µL 2CD10°
  55,5µL H2O
  
  PCR parameter:
  92°C 1min
  35 cycles : 92°C 10s, 66°C 30s, 72°C 10s
  End at 4°C
  Gel 3% 45min 75V 10µL + 1,1µL
  

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