Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/06

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(Autocreate 2012/12/06 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes)
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==Entry title==
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==December 6, 2012==
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* Insert content here...
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'''Assemblies'''
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* Final Gibson assembly products
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# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
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# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
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'''Dephosphorylation''' (11/23/12)
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> Dephosphorylation (Roche)
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# pSB1A3 - X/S = 2000 bp
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{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
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|-
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| <u>Reagent</u> || <u>Volume</u>
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|-
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| DNA (clean digest) || 11.0 (~200 ng)
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|-
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| 10x phos. buffer || 1.5
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|-
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| phosphatase || 0.5
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|-
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| dH<sub>2</sub>O || 2.0
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|-
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| &nbsp; || 15 μL
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|}
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* Incubate at 37°C/ 10 min.
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* Heat inactivate at 75°C/ 2 min.
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* [final] = 200 ng/μL / 15 μL = ~13 ng/μL
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{| class="wikitable" width=700px
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| &nbsp; || H2B || LOV  || Negative Control || H2B || LOV  || Negative Control
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|-
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| Insert DNA (2x mol vector) || 1.0 μL || 1.0 || --- || 1.0 || 1.0 || ---
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|-
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| Vector DNA (50 ng) || 1.5  || 1.5 || 1.5 || 1.5  || 1.5 || 1.5
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|-
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| 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0
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|-
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| T4 ligase || 1.0  NEB || 1.0 NEB || 1.0 NEB || 1.0 Roche || 1.0 Roche || 1.0 Roche
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|-
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| dH<sub>2</sub>O || 1.5 || 1.5 || 2.5 || 1.5 || 1.5 || 2.5
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|-
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| TOTAL || 10.0 || 10.0 || 10.0  || 10.0 || 10.0 || 10.0
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|-
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| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes.
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|}
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Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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Revision as of 20:40, 6 December 2012

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December 6, 2012


Assemblies

  • Final Gibson assembly products
  1. pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
  2. pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end

Dephosphorylation (11/23/12)

> Dephosphorylation (Roche)

  1. pSB1A3 - X/S = 2000 bp
Reagent Volume
DNA (clean digest) 11.0 (~200 ng)
10x phos. buffer 1.5
phosphatase 0.5
dH2O 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


  H2B LOV Negative Control H2B LOV Negative Control
Insert DNA (2x mol vector) 1.0 μL 1.0 --- 1.0 1.0 ---
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 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
dH2O 1.5 1.5 2.5 1.5 1.5 2.5
TOTAL 10.0 10.0 10.0 10.0 10.0 10.0
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes.

Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101




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