Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/03/13: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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| colspan="2"| | | colspan="2"| | ||
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| ATP || 10 mM || 10.0 | | ATP || 10 mM || 10.0 | ||
|- | |||
| dH<sub>2</sub>O || || 490.0 | |||
|- | |- | ||
| Final vol. || || 1000 uL | | Final vol. || || 1000 uL | ||
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* Golden Gate assembly reactions | * 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 --> | {| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| Reagent || Promega (1 | | Reagent || Promega (1) || Promega (2) || Roche (9, 10) || Roche (11, 12) | ||
|- | |- | ||
| gg2 pSB1A3* | | gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| gg3 hPCD* | | gg3 hPCD* || 1.0 || --- || 1.0 || --- | ||
|- | |- | ||
| gg4 BL01* | | gg4 BL01* || 1.0 || --- || 1.0 || --- | ||
|- | |- | ||
| ligase buffer | | ligase buffer || 1.0 || 1.0|| 5.0 || 5.0 | ||
|- | |- | ||
| NEB T4 ligase | | NEB T4 ligase || 0.25 || 0.25 || 0.25 || 0.25 | ||
|- | |- | ||
| NEB BsmBI | | NEB BsmBI || 0.5 || 0.5 || 0.5 || 0.5 | ||
|- | |- | ||
| dH<sub>2</sub>O | | 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 | Note: *DNA is 20 fmole/μL | ||
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* Transformations | * 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 | ||
Latest revision as of 22:32, 26 September 2017
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03/13/13
Golden Gate Assembly
Note: *DNA is 20 fmole/μL Thermal cycler
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