Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/21: Difference between revisions
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* Run entire 30 μL on a 1% gel (use the big fat-tooth well comb) | * Run entire 30 μL on a 1% gel (use the big fat-tooth well comb) | ||
* Cut band out of gel (use UV box) | * Cut band out of gel (use UV box) | ||
Concentrations: | |||
... | |||
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# H2B = 410 | # H2B = 410 | ||
# LOV = 461 | # LOV = 461 | ||
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| <u>Reagent</u> || <u>Volume</u> | | <u>Reagent</u> || <u>Volume</u> | ||
|- | |- | ||
| DNA (clean digest) || up to | | DNA (clean digest) || up to 11.0 (~200 ng) | ||
|- | |- | ||
| 10x buffer d.p. || | | 10x buffer d.p. || 1.5 | ||
|- | |- | ||
| phosphatase || | | phosphatase || 0.5 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 2.0 | ||
|- | |- | ||
| || | | || 15 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~13 ng/μL | ||
|} | |} | ||
* Heat inactivate .... | |||
'''Ligation''' | '''Ligation''' | ||
* Use 20 ng of vector for each ligation = '''1 μL pSB1A3''' | * Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3''' | ||
* Use 2x moles of H2B insert, relative to vector: xμL H2B insert = | * Use 2x moles of H2B insert, relative to vector: xμL H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / ### ng/μL H2B = '''1.28 μL H2B''' | ||
* Use 2x moles of LOV insert, relative to vector: xμL LOV insert = | * Use 2x moles of LOV insert, relative to vector: xμL LOV insert = 3 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / ### ng/μL LOV = '''0.8 μL LOV''' | ||
{| class="wikitable" width=400px | {| class="wikitable" width=400px | ||
| || H2B || LOV || Negative Control | | || H2B || LOV || Negative Control | ||
|- | |- | ||
| Insert DNA (2x mol vector) || | | Insert DNA (2x mol vector) || ### μL || ### || --- | ||
|- | |- | ||
| Vector DNA (50 ng) || 1. | | Vector DNA (50 ng) || 1.5 || 1.5 || 1.5 | ||
|- | |- | ||
| 2x Roche Rapid Ligation buffer || | | 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 | ||
|- | |- | ||
| New England Biolabs T4 ligase || 1.0 || 1.0 || 1.0 | | New England Biolabs T4 ligase || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || ### || ### || ### | ||
|- | |- | ||
| TOTAL || | | TOTAL || 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 14:14, 21 November 2012
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11/21/12
PCR program:
Results of the PCR products, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.
Assemblies
Concentrations: ...
Dephosphorylation > Dephosphorylation (Roche)
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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