Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/21: Difference between revisions
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*'''[[User:Karmella Haynes|---Karmella]] 15:56, 21 November 2012 (EST)''': Digest, purification, and ligation | *'''[[User:Karmella Haynes|---Karmella]] 15:56, 21 November 2012 (EST)''': Digest, purification, and ligation | ||
'''Assemblies''' | |||
# | |||
'''Ligation''' | |||
* Use 20 ng of vector for each ligation = '''1 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) || 1.0 || 1.0 || 1.0 | |||
|- | |||
| 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 || 4.0 || 4.0 || 5.0 | |||
|- | |||
| 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 | |||
Revision as of 13:59, 21 November 2012
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11/21/12
PCR program:
Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.
Assemblies Ligation
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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