User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/15: Difference between revisions
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9, 10. Roche - hPCD+BL01+pSB1A3<br> | 9, 10. Roche - hPCD+BL01+pSB1A3<br> | ||
11, 12. Roche - pSB1A3<br> | 11, 12. Roche - pSB1A3<br> | ||
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| Reagent || Promega (1,2) || Promega (3, 4) || NEB (5, 6) || NEB (7, 8) || Roche (9, 10) || Roche (11, 12) | | 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 | | gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| 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 || --- | ||
|- | |- | ||
| | | ligase buffer || 1.0 || 1.0|| 1.0 || 1.0|| 5.0 || 5.0 | ||
|- | |- | ||
| NEB T4 | | NEB T4 ligase || 0.25 || 0.25 || 0.25 || 0.25 || 0.25 || 0.25 | ||
|- | |- | ||
| BsmBI || 0.5 || 0.5 | | NEB 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 | ||
|- | |- | ||
| || 10.0 || 10.0 | | || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 | ||
|} | |} | ||
Note: *DNA is 20 fmole/μL | |||
Thermal cycler | Thermal cycler | ||
Line 64: | Line 63: | ||
* 60°C, 20 min. | * 60°C, 20 min. | ||
* 80°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 | |||
Revision as of 18:12, 16 January 2013
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01/15/13
Golden Gate Assembly optimization
1, 2. Promega - hPCD+BL01+pSB1A3
Note: *DNA is 20 fmole/μL Thermal cycler
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