Haynes Lab:Notebook/Synthetic Chromatin for Cancer Research/2015/06/15

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6-4-15


David Tze - assemblies: linkers + hPCD

  • Assemblies
  1. DT009_pUC57: flyPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
  2. DT0010_pUC57: flyPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
  3. DT0011_pUC57: flyPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
  4. DT0012_pUC57: flyPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931


  • Digests (Fermentas FD)
Reagent Rxn1 Expected bands:
1. fshPCD(E/S) = 3200, 186*

(*) Bands that were cut out for purification

30 μL/lane, 1% agarose; Ladder
    • Well 1 was punctured so the ladder was put into Well 3 as well for safe measures.
DNA (plasmid) 25.0
10x buffer 3.0
EcoRI 1.0
enzyme 2 1.0
dH2O ---
  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. flyPCD(E/S) --- --- ---
2. PLflex_pUC57(E/X) --- --- ---
3. PLflex4_pUC57(E/X) --- --- ---
4. PLrigid_pUC57(E/X) --- --- ---
5. PLrigid4_pUC57(E/X) --- --- ---
    • Readings were odd, which is typical for the Sigma gel extraction products.
    • Will use an arbitrary volume of 2.0 insert and 2.0 vector as it has worked before.
    • Good insert : vector ratio should not be difficult to achieve since insert is very short.
    • Linkers were extracted in a previous experiment. Concentrations were taken from that entry as the same linkers were used.


  • Ligations
    • 2:1 ratio calculation: 186 bp insert / ~2800 bp vector * 2 * 50 = 6.64 ng insert
  1. DT009_pUC57: fshPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
  2. DT0010_pUC57: fshPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
  3. DT0011_pUC57: fshPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
  4. DT0012_pUC57: fshPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931
  5. PLflex_pUC57(E/X)/2763
  • Note: Used one negative (one vector only)
Reagent Rxn1-4 Rxn5
Insert DNA 2.0 ---
Vector DNA 2.0 2.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 2.0
  10.0 μL 10.0 μL


RESULTS

  • Looks successful: ~200 colonies on the ligation plates, 1 colony on the neg. ctrl plate.
  • Picked two colonies from each plate (1-4) for streak library and 5 mL cultures



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