User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/15

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01/15/13

  • Golden gate assembly: test different ligase buffers


Golden Gate Assembly optimization

  • Test different buffers
Buffer Tris-HCl MgCl2 DTT ATP
10x Promega 300 mM (pH 7.8) 100 mM 100 mM 10 mM
10x NEB 500 mM (pH 7.5) 100 mM 100 mM 10 mM
2x Roche  ?  ?  ?  ?
  • Make Promega buffer from scratch.


  • Golden Gate assembly reactions

1, 2. Promega - hPCD+BL01+pSB1A3
3, 4. Promega - pSB1A3
5, 6. NEB - hPCD+BL01+pSB1A3
7, 8. NEB - pSB1A3
9, 10. Roche - hPCD+BL01+pSB1A3
11, 12. Roche - pSB1A3

Reagent Promega (1,2) Promega (3, 4) NEB (5, 6) NEB (7, 8) Roche (9, 10) Roche (11, 12)
gg2 pSB1A3* 1.0 1.0 1.0 1.0 1.0 1.0
gg3 hPCD* 1.0 --- 1.0 --- 1.0 ---
gg4 BL01* 1.0 --- 1.0 --- 1.0 ---
ligase buffer 1.0 1.0 1.0 1.0 5.0 5.0
NEB T4 ligase 0.25 0.25 0.25 0.25 0.25 0.25
NEB BsmBI 0.5 0.5 0.5 0.5 0.5 0.5
dH2O 5.25 7.25 5.25 7.25 1.25 3.25
  10.0 10.0 10.0 10.0 10.0 10.0

Note: *DNA is 20 fmole/μL

Thermal cycler

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


  • Transformations
    • All odd samples: 50 μL DH5α-Turbo; ice 5 min.; plate on amp agar
    • All even samples: 50 μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar



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