From 2014.igem.org

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22^nd August

• Chewback : purification with Qiagen Kit, dosage on nanodrop
  
  ELP (6240pb) 8,2 ng/μl
  RFP (706pb) 12,8ng/μl
  
  
  We want to use 25 fmol => we use promega website (μg to pmol converter)
  
  ELP = 0,002pmol/μl => 25 fmol = 12,5μl
  RFP = 0,027pmol/μl => 25 fmol = 0,9μl
  
  Do the mix on ice :
  

  RFP	0,9μl
  Plasmid	12,5μl
  Master Mix	12,5μl

  
  Inoculate immediately 1h at 50°C
  

  • Transformation of competent cells and spread on LB+Amp plates
  • Gel 1% (30min, 100V)


• Biobrick assembly
  • Digestion
  Dilution ELP 2,1μl +7,7μl H2O
  Dilution SLP 3,6μl +6,4μl H2O
  Dilution RLP 1,97μl +8,03μl H2O
  
  
  incubation 1h30 at 37°C :
  

  Enzyme 1 (Nhe1 or X)	0,5μl
  Enzyme 2 (S)	0,5lμl
  10X Buffer 4	5μl
  DNA	10μl
  H2O	qsp 50μl

  - Enzyme inactivated 20min at 80°C
  - Gel 1% (30min, 100V)
  
  
  • Ligation

  Plasmid	4μl
  Insert	4μl
  Buffer 10X	1μl
  Taq ligase	0,5μl
  H2O	qsp 10μl

  => 30min at 16°C
  
  
  • Transformation

• We change the water of dialysis at 9h.