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 | |||
| Reagent || Conc. || Volume | |||
|- | |||
| 1M Tris-HCl (pH 7.8) || 300 mM || 300.0 μL | |||
|- | |||
| 1M MgCl<sub>2</sub> || 100 mM || 100.0 | |||
|- | |||
| 1M DTT || 100 mM || 100.0 | |||
|- | |||
| ATP || 10 mM || 10.0 | |||
|- | |||
| dH<sub>2</sub>O || || 490.0 | |||
|- | |||
| Final vol. || || 1000 uL | |||
|} | |||
* Golden Gate assembly reactions | |||
# Promega - H2B + LOV-his + pSB1A3 | |||
# Promega - pSB1A3 | |||
# Roche - H2B + LOV-his + pSB1A3 | |||
# Roche - pSB1A3 | |||
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | |||
|- valign="top" | |||
| Reagent || Promega (1) || Promega (2) || Roche (9, 10) || Roche (11, 12) | |||
|- | |||
| gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| gg3 hPCD* || 1.0 || --- || 1.0 || --- | |||
|- | |||
| gg4 BL01* || 1.0 || --- || 1.0 || --- | |||
|- | |||
| ligase buffer || 1.0 || 1.0|| 5.0 || 5.0 | |||
|- | |||
| NEB T4 ligase || 0.25 || 0.25 || 0.25 || 0.25 | |||
|- | |||
| NEB BsmBI || 0.5 || 0.5 || 0.5 || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 5.25 || 7.25 || 1.25 || 3.25 | |||
|- | |||
| || 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 | |||
** 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 21:31, 12 March 2013
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03/13/13
Golden Gate Assembly
Note: *DNA is 20 fmole/μL Thermal cycler
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