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

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Line 47: Line 47:
| gg3 hPCD || 1.0 || --- || 1.0 || --- || 1.0 || ---  
| gg3 hPCD || 1.0 || --- || 1.0 || --- || 1.0 || ---  
|-
|-
-
| gg4 BL01 || 1.0 || ---  
+
| gg4 BL01 || 1.0 || --- || 1.0 || --- || 1.0 || ---  
|-
|-
-
| 10x ligase buffer || 1.0 || 1.0
+
| 10x ligase buffer || 1.0 || 1.0|| 1.0 || 1.0|| 5.0 || 5.0
|-
|-
-
| NEB T4 lgase || 0.25 || 0.25
+
| NEB T4 lgase || 0.25 || 0.25 || 0.25 || 0.25 || 0.25 || 0.25
|-
|-
-
| BsmBI || 0.5 || 0.5
+
| BsmBI || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5
|-
|-
-
| dH<sub>2</sub>O || 5.25 || 7.25
+
| dH<sub>2</sub>O || 5.25 || 7.25 || 5.25 || 7.25 || 1.25 || 3.25
|-
|-
-
| &nbsp; || 10.0 || 10.0
+
| &nbsp; || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 || 10.0
|}
|}

Revision as of 21:07, 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 --- 1.0 --- 1.0 ---
10x ligase buffer 1.0 1.0 1.0 1.0 5.0 5.0
NEB T4 lgase 0.25 0.25 0.25 0.25 0.25 0.25
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

Thermal cycler

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




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