Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/06: Difference between revisions
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(Autocreate 2012/12/06 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes) |
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== | ==December 6, 2012== | ||
---- | |||
'''Assemblies''' | |||
* Final Gibson assembly products | |||
# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end | |||
# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end | |||
'''Dephosphorylation''' (11/23/12) | |||
> Dephosphorylation (Roche) | |||
# pSB1A3 - X/S = 2000 bp | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table --> | |||
|- | |||
| <u>Reagent</u> || <u>Volume</u> | |||
|- | |||
| DNA (clean digest) || 11.0 (~200 ng) | |||
|- | |||
| 10x phos. buffer || 1.5 | |||
|- | |||
| phosphatase || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 2.0 | |||
|- | |||
| || 15 μL | |||
|} | |||
* Incubate at 37°C/ 10 min. | |||
* Heat inactivate at 75°C/ 2 min. | |||
* [final] = 200 ng/μL / 15 μL = ~13 ng/μL | |||
'''Ligation''' | |||
* Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3''' | |||
* Use 1x moles of H2B insert, relative to vector: 1xμL H2B insert = 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 60 ng/μL H2B = at least '''0.07 μL H2B''' | |||
* Use 1x moles of LOV insert, relative to vector: 1xμL LOV insert = 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 62 ng/μL LOV = at least '''0.07 μL LOV''' | |||
* Since the calulated amounts are so small and a volume of 5 μL is needed to add to the Gibson assembly mix, each value was multiplied by 2 and rounded for simplicity | |||
{| class="wikitable" width=700px | |||
| || H2B-LOV || LOV-H2B || Negative Control | |||
|- | |||
| H2B-LOV H2B insert DNA (1x mol vector) || 1.0 μL || 0 || 0 | |||
|- | |||
| H2B-LOV LOV insert DNA (1x mol vector) || 1.0 μL || 0 || 0 | |||
|- | |||
| LOV-H2B H2B insert DNA (1x mol vector) || 0 μL || 1.0 || 0 | |||
|- | |||
| LOV-H2B LOV insert DNA (1x mol vector) || 0 μL || 1.0 || 0 | |||
|- | |||
| Vector DNA (39 ng) || 3.0 || 3.0 || 3.0 | |||
|- | |||
| Gibson master mix || 15.0 || 15.0 || 15.0 | |||
|- | |||
| Total volume || 20.0 || 20.0 || 18.0 | |||
|- | |||
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 50°C for 1 hour. | |||
|} | |||
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101 | |||
[[Image:VN_Gibson_Assembly_Trial_1.png|thumb|frame|center|No colonies were seen the next day.]] | |||
'''Made more pSB1A3 stock''' | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
|Trial || 260 || 280 || 260/280 || ng/μL | |||
|- | |||
|1 || 0.062|| 0.035 || 1.773|| 62.46 | |||
|- | |||
|2 || 0.052|| 0.03 || 1.722|| 52.032 | |||
|- | |||
|3 || 0.082|| 0.047|| 1.762|| 82.222 | |||
|- | |||
|} | |||
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Revision as of 05:37, 18 April 2013
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December 6, 2012Assemblies
Dephosphorylation (11/23/12) > Dephosphorylation (Roche)
Ligation
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101 Made more pSB1A3 stock
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