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

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05/28/15

  • David Tze - assemblies: linkers + hPCD
  • Ben - DBN007 5 mL cultures


David Tze - assemblies: linkers + hPCD

  • Assemblies
  1. DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
  2. DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
  3. DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
  4. DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931


  • Digests (Fermentas FD)
Reagent Rxn1 Rxn2-5 Expected bands:
1. hPCD(E/S) = 186
2. PLflex_pUC57(E/X) = 2763
3. PLflex4_pUC57(E/X) = 2930
4. PLrigid_pUC57(E/X) = 2763
5. PLrigid4_pUC57(E/X) = 2931 		Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 25.0 15.0
10x buffer 3.0 3.0
enzyme 1 1.0 1.0
enzyme 2 1.0 1.0
dH2O --- 10.0
  30 μL

--> 37°C/ ~30 min.


  • Gel purification
    • Sigma GenElute Gel purification kit
    • Elute & back-elute w/ 25 μL elution sln.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. hPCD(E/S) --- --- ---
2. PLflex_pUC57(E/X) --- --- ---
3. PLflex4_pUC57(E/X) --- --- ---
4. PLrigid_pUC57(E/X) --- --- ---
5. PLrigid4_pUC57(E/X) --- --- ---


  • Ligations
    • 2:1 ratio calculation: 186 bp insert / ~2800 bp vector * 2 * 50 = 6.64 ng insert
  1. DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
  2. DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
  3. DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
  4. DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931
  5. PLflex_pUC57(E/X)/2763
Reagent Rxn1-4 Rxn5
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL



  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.


  • Digests
    • Cut w/ E/S
Reagent Volume Expected:
1-2. DBN007_pSB1A3 = 2197, 2132
DBN001 = 2132, 224
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
SpeI 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~15 min.





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