Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/14: Difference between revisions
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| LOV|| 0.011|| 0.004 || 2.769 || 11.492 | | LOV|| 0.011|| 0.004 || 2.769 || 11.492 | ||
|- | |- | ||
| pSB1A3|| 0.004 || 0.001 || 2.857 | 4.233 | | pSB1A3|| 0.004 || 0.001 || 2.857 || 4.233 | ||
|} | |} | ||
---- | |||
*'''[[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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Revision as of 19:48, 15 November 2012
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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 |