Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/14: 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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*'''[[User:Karmella Haynes|---Karmella]] 21:34, 15 November 2012 (EST)''': Next step - ligation & transformation<br> | |||
Refer to this page for a full description: http://openwetware.org/wiki/Haynes:Assembly101 | |||
* Use 20 ng of vector for each ligation = '''5 μL pSB1A3''' | |||
* Use 2x moles of H2B insert, relative to vector: xμL H2B insert = 2 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 6.384 ng/μL H2B = '''1.28 μL H2B''' | |||
* Use 2x moles of LOV insert, relative to vector: xμL LOV insert = 2 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 11.492 ng/μL LOV = '''0.8 μL LOV''' | |||
{| class="wikitable" width=400px | |||
| || H2B || LOV || Negative Control | |||
|- | |||
| Insert DNA (2x mol vector) || 1.0 μL || 1.0 || 0 | |||
|- | |||
| Vector DNA (50 ng) || 5.0 || 5.0 || 5.0 | |||
|- | |||
| 2x Roche Rapid Ligation buffer || 7.0 || 7.0 || 7.0 | |||
|- | |||
| New England Biolabs T4 ligase || 1.0 || 1.0 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 0 || 0 || 1.0 | |||
|- | |||
| TOTAL || 14.0 || 14.0 || 14.0 | |||
|- | |||
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes. | |||
|} | |||
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101 | |||
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Latest revision as of 22:15, 26 September 2017
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November 14, 2012
Refer to this page for a full description: http://openwetware.org/wiki/Haynes:Assembly101
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101 |