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

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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

No colonies were seen the next day.
No colonies were seen the next day.

Made more pSB1A3 stock

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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