User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/15: Difference between revisions

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* Make Promega buffer from scratch. Test Promega and NEB formulas.
* Make Promega buffer from scratch.
* Follow same procedure as ###
 
* Transform into ### cells
 
* Golden Gate assembly reactions
1, 2. Promega - hPCD+BL01+pSB1A3<br>
3, 4. Promega - pSB1A3<br>
5, 6. NEB - hPCD+BL01+pSB1A3<br>
7, 8. NEB - pSB1A3<br>
9, 10. Roche - hPCD+BL01+pSB1A3<br>
11, 12. Roche - pSB1A3<br>
 
 
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
|- valign="top"
| 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
|-
| dH<sub>2</sub>O || 5.25 || 7.25
|-
| &nbsp; || 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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Revision as of 17:46, 16 January 2013

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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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