Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/03/13

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03/13/13

  • Golden Gate assembly



Golden Gate Assembly

  • First, make fresh 10x Promega Buffer:
Reagent Conc. Volume
1M Tris-HCl (pH 7.8) 300 mM 300.0 μL
1M MgCl2 100 mM 100.0
1M DTT 100 mM 100.0
ATP 10 mM 10.0
dH2O 490.0
Final vol. 1000 uL


  • Golden Gate assembly reactions
  1. Promega - H2B + LOV-his + pSB1A3
  2. Promega - pSB1A3
  3. Roche - H2B + LOV-his + pSB1A3
  4. Roche - pSB1A3

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