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

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==Entry title==
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==December 6, 2012==
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----
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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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'''Ligation'''
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* Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3'''
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* 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'''
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* 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'''
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* 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
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{| class="wikitable" width=700px
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| &nbsp; || H2B-LOV || LOV-H2B  || Negative Control
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|-
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| H2B-LOV H2B insert DNA (1x mol vector) || 1.0 μL || 0 || 0
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|-
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| H2B-LOV LOV insert DNA (1x mol vector) || 1.0 μL || 0 || 0
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|-
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| LOV-H2B H2B insert DNA (1x mol vector) || 0 μL || 1.0 || 0
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|-
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| LOV-H2B LOV insert DNA (1x mol vector) || 0 μL || 1.0 || 0
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|-
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| Vector DNA (39 ng) || 3.0  || 3.0 || 3.0
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|-
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| Gibson master mix || 15.0 || 15.0 || 15.0
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|-
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| Total volume || 20.0 || 20.0 || 18.0
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|-
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| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 50°C for 1 hour.
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|}
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Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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[[Image:VN_Gibson_Transformation_Trial_1.png‎|thumb|frame|center|No colonies were seen the next day.]]
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Revision as of 10:04, 8 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

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
  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
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at 50°C for 1 hour.

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

Image:VN Gibson Transformation Trial 1.png
No colonies were seen the next day.



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