User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/13

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05/13/15

  • Rene - PCR clone library
  • Ryan - cultures
  • Cas-tone - cultures from streaks; Addgene plasmids, 5mL each, overnight; 7 total (4 amp, 3 kan)



Rene - PCR clone library

  • Use the CloneJET PCR Cloning Kit - MAN0012707 CloneJET PCR Cloning (manual)
    • pJET1.2/blunt vector is 5'-phosphorylated and accepts blunt products (Phusion PCR)
    • All common laboratory E. coli strains can be directly transformed with the ligation product
    • Re-circularized vector expresses toxic enzyme, no blue/white screening needed
    • Optimal insert : vector (0.05 pmol ends) ratio is 3:1; Calculation: length of insert DNA x 0.05 = ng insert to use


  • Ligations - CloneJET PCR Cloning Kit
  1. Luc14 + CRISPR g034; size = 630
  2. Gal4EED/luc + CRISPR g034 = 630


Reagent Volume
2x reaction buffer 10.0
PCR product (31.5 ng) 2.0
pJET1.2/blunt vector 1.0
T4 ligase 1.0
dH2O 6.0
  20.0 μL
  • Incubate the ligation mixture at room temperature (22°C) for 5 min.
  • Transform DH5α-turbo with 10 μL ligation reaction. Follow quick protocol.
  • Plate on 100 μg/mL amp.


RESULTS (5/14/15)

  • Worked, but not very many colonies
  • Redo ligation with 2x as much insert
  • Follow long transformation protocol (heat-shock & recovery in SOC)