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

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* Make Promega buffer from scratch. Test Promega and NEB formulas.
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* Make Promega buffer from scratch.
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* Follow same procedure as ###
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* Transform into ### cells
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* Golden Gate assembly reactions
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1, 2. Promega - hPCD+BL01+pSB1A3<br>
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3, 4. Promega - pSB1A3<br>
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5, 6. NEB - hPCD+BL01+pSB1A3<br>
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7, 8. NEB - pSB1A3<br>
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9, 10. Roche - hPCD+BL01+pSB1A3<br>
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11, 12. Roche - pSB1A3<br>
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{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
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|- valign="top"
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| Reagent || Promega (1,2)  || Promega (3, 4) || NEB (5, 6) || NEB (7, 8) || Roche (9, 10) || Roche (11, 12)
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|-
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| gg2 pSB1A3 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0
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|-
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| gg3 hPCD || 1.0 || --- || 1.0 || --- || 1.0 || ---
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|-
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| gg4 BL01 || 1.0 || ---
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|-
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| 10x ligase buffer || 1.0 || 1.0
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|-
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| NEB T4 lgase || 0.25 || 0.25
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|-
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| BsmBI || 0.5 || 0.5
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|-
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| dH<sub>2</sub>O || 5.25 || 7.25
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|-
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| &nbsp; || 10.0 || 10.0
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|}
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Thermal cycler
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* [45°C, 2 min.; 16°C, 5 min.] x25
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* 60°C, 20 min.
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* 80°C, 20 min.
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Revision as of 19:46, 16 January 2013

Karmella's BioBrick Cloning Main project page
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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 ---
10x ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

Thermal cycler

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




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