User:Cassandra M Barrett/Notebook/Open Chromatin/2015/11/06

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LCR pJET

Purpose: to verify that LCR can be successfully performed on two linear parts. Parts will be fused using LCR and ligated into a pJET vector. Ligation product will be transformed into 'E. coli' and colony PCR (and possibly sequencing) will be performed to confirm properly assembled insert (pJET has sequencing primers already designed for just outside of insertion point)

Methods:

Perform LCR on Gal4DB_mCh and ATF2 parts

Both parts have already been phosphorylated and diluted to 30nM (9/6). 300nM bridge was used (newly ordered). Sequence: 5'- ATG GAC GAG CTG TAC AAG GAG ATG ACA CTG AAA TTT GGT CCA - 3' TM= 66.4C. New bridge was reconstituted to 100uM stock concentration from which a 300nM stock was created.

  • Ampligase 1uL
  • Ampligase Buffer (10X) 1uL
  • DMSO (8%v/v) 0.8uL
  • Betaine (.45M final) 0.8uL
  • bridge (30nM) 2uL
  • Gal4DB_mCh (phosphorylated, 3nM) 2uL
  • ATF2 (phosphorylated, 3nM) 2uL
  • Water 0.4uL

Cycle with the following parameters:

  • 2 min at 94C
  • 50 cycles(10s at 94C, 30s at 55C, 60s at 66C)
  • 4C hold

pJET Cloning

    • pJET1.2/blunt vector is 5'-phosphorylated and accepts blunt products
    • 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 & transformations - CloneJET PCR Cloning Kit


Reagent Volume
2x Roche lig buf 5.0
LCR product 1.0
pJET1.2/blunt vector 1.0
NEB T4 ligase 1.0
dH2O 2.0
  10.0 μL
  • Set up two ligations, one with LCR product and the other with water as a control
  • Incubate the ligation mixtures at room temperature (22°C) for 5 min.
  • Transform 50 μL DH5α-turbo with 10 μL ligation reaction.

Follow standard DH5alpha transformation protocol:

  • Add 50uL of DH5alpha turbo cells to each tube with appropriate sample (10uL LCR pJET lig, 10uL water ligation control and 10 of water as +control)
  • Incubate on ice for 30min
  • Heat shock at 42C for 35sec
  • Incubate on ice for 2min
  • Add 750uL of SOC media (RT) to each tube
  • Shake 37C for 1hr
  • Plate on LB+AMP (Spin down, remove 200uL of supernatant, resuspend and plate 100uL)
  • Incubate overnight at 37C



Results 11/9/15

No growth on any plates =( Possible failed transformation/bad cell batch/ mislabeled antibiotic plates. Will Reattempt entire LCR pJET trouble shooting experiment. Next time add positive transformation control with MV10 vector to ensure that cells are good.