User:Karmella Haynes/Notebook/BioBrick cloning/2013/08/29

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08/29/13

  • Golden Gate Assembly optimization - increase ligase amount; redo last successful hPCD assembly



Golden Gate

  • Try to increase robustness by using more T4 ligase...
  1. hPCD + BL01 + pSB1A3
  2. pSB1A3 alone (neg ctrl)
  • GG-PCR fragments were generated on 1/10/13 and diluted to 20 fmol/μL
  • Try the Promega formula home-made buffer (worked last time)


  • Golden Gate assembly reactions

1, 2. 0.25 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3
1, 2. 1.0 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3


Reagent Original protocol Original protocol More ligase More ligase H2B+LOV
gg2 pSB1A3* 1.0 1.0 1.0 1.0 1.0
gg3 hPCD* 1.0 --- 1.0 --- 1.0 H2B gg
gg4 BL01* 1.0 --- 1.0 --- 1.0 LOV gg
10x Promega ligase buffer 1.0 1.0 1.0 1.0 1.0
NEB T4 ligase 0.25 0.25 1.0 1.0 1.0
NEB BsmBI 0.5 0.5 0.5 0.5 0.5
dH2O 5.25 7.25 4.5 6.5 4.5
  10.0 10.0 10.0 10.0 10.0

Note: *DNA is 20 fmol/μL

GG Thermal cycling

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.
  • 4°C, ∞


  • Transformations (08/30/13)
    • 40 μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 750 μL SOC medium; shake @ 37°C 30 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar
  • Results
    • No colonies on any plates. Start again from generation of PCR fragments. Try DpnI clean-up approach next time.



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