Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/03/13: Difference between revisions
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(Autocreate 2013/03/13 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes) |
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== | ==03/13/13== | ||
* | * Golden Gate assembly | ||
---- | |||
'''Golden Gate Assembly'''<br> | |||
* First, make fresh 10x Promega Buffer: | |||
{| {{table}} border="1" cellspacing="3" | |||
|- valign="top" bgcolor=#cfcfcf | |||
| Buffer || Tris-HCl || MgCl<sub>2</sub> || 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<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 || --- || 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 | |||
|- | |||
| 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 | |||
|} | |||
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 | |||
Revision as of 19:00, 12 March 2013
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03/13/13
Golden Gate Assembly
1, 2. Promega - hPCD+BL01+pSB1A3
Note: *DNA is 20 fmole/μL Thermal cycler
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