User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/28

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05/28/15

  • David Tze - assemblies: linkers + hPCD


David Tze - assemblies: linkers + hPCD

  • Assemblies
  1. DT001 hPCD(E/S) + PLflex_pUC57(E/X)
  2. DT002 hPCD(E/S) + PLflex4_pUC57(E/X)
  3. DT003 hPCD(E/S) + PLrigid_pUC57(E/X)
  4. DT004 hPCD(E/S) + PLrigid4_pUC57(E/X)
  1. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


  • Digests (Fermentas FD)
    • Specific notes
Reagent Volume
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
  1. insert/(a/b)/size + vector/(c/d)/size
  2. vector/(c/d)/size
Reagent Rxn1 Rxn2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL




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