Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/21: Difference between revisions
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| DNA (clean digest) || 11.0 (~200 ng) | | DNA (clean digest) || 11.0 (~200 ng) | ||
|- | |- | ||
| 10x buffer | | 10x phos. buffer || 1.5 | ||
|- | |- | ||
| phosphatase || 0.5 | | phosphatase || 0.5 | ||
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'''Ligation''' | '''Ligation''' | ||
* Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3''' | * Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3''' | ||
* Use | * Use 3x moles of H2B insert, relative to vector: xμL H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 16 ng/μL H2B = at least '''0.8 μL H2B''' | ||
* Use | * Use 3x moles of LOV insert, relative to vector: xμL LOV insert = 3 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 25 ng/μL LOV = at least '''0.5 μL LOV''' | ||
{| class="wikitable" width= | {| class="wikitable" width=700px | ||
| || H2B || LOV || Negative Control | | || H2B || LOV || Negative Control || H2B || LOV || Negative Control | ||
|- | |- | ||
| Insert DNA (2x mol vector) || | | Insert DNA (2x mol vector) || 1.0 μL || 1.0 || --- || 1.0 || 1.0 || --- | ||
|- | |- | ||
| Vector DNA (50 ng) || 1.5 || 1.5 || 1.5 | | Vector DNA (50 ng) || 1.5 || 1.5 || 1.5 || 1.5 || 1.5 || 1.5 | ||
|- | |- | ||
| 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 | | 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 | ||
|- | |- | ||
| | | T4 ligase || 1.0 NEB || 1.0 NEB || 1.0 NEB || 1.0 Roche || 1.0 Roche || 1.0 Roche | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 1.5 || 1.5 || 2.5 || 1.5 || 1.5 || 2.5 | ||
|- | |- | ||
| TOTAL || 10.0 || 10.0 || 10.0 | | TOTAL || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 | ||
|- | |- | ||
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes. | | colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes. |
Revision as of 14:58, 23 November 2012
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
11/21/12Results from yesterday's ligation
PCR program:
Assemblies
> Dephosphorylation (Roche)
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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