From 2014.igem.org

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27^th August

• 3 digestions (ELP, SLP and RLP)
  We want remove the stop codon (with Nhe1, Spe1 and dephosphorylation) and recover our insert (ELp, SLP and RLP)
  
  Dilution DNA :
  - ELP : 328,5 ng/μl => 3μl + 7μl H2O
  - SLP : 266,4 ng/μl => 3,75μl + 6,25μl H2O
  - RLP: 429,9 ng/μl => 2,33 μl + 7,67μl H2O
  

  Buffer 4 10X	5μl
  Enzyme 1	1μl
  Enzyme 2	1μl
  DNA	10μl
  H2O	qsp 50μl

  
  => Gel


• Ligation (assembly)


• Digestion of pSB1C3
  Dilution DNA: pSB1C3: 224,2 ng/μl  4,46μl + 5,54 H2O
  Test with 1,5μl, 1μl, 0,5μl and 0μl of enzyme
  Gel


• Ligation (pSB1C3)

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

  => 30min at 45°C and transformation