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

(Difference between revisions)
 Revision as of 19:40, 6 December 2012 (view source) (→Entry title)← Previous diff Revision as of 08:59, 8 December 2012 (view source) (→December 6, 2012)Next diff → Line 38: Line 38: * [final] = 200 ng/μL / 15 μL = ~13 ng/μL * [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 {| class="wikitable" width=700px - |   || H2B || LOV  || Negative Control || H2B || LOV || Negative Control + |   || 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 |- |- - | Insert DNA (2x mol vector) || 1.0 μL || 1.0 || --- || 1.0 || 1.0 || --- + | LOV-H2B H2B insert DNA (1x mol vector) || 0 μL || 1.0 || 0 |- |- - | Vector DNA (50 ng) || 1.5  || 1.5 || 1.5 || 1.5  || 1.5 || 1.5 + | LOV-H2B LOV insert DNA (1x mol vector) || 0 μL || 1.0 || 0 |- |- - | 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 + | Vector DNA (39 ng) || 3.0 || 3.0 || 3.0 |- |- - | T4 ligase || 1.0 NEB || 1.0 NEB || 1.0 NEB || 1.0 Roche || 1.0 Roche || 1.0 Roche + | Gibson master mix || 15.0 || 15.0 || 15.0 - |- + - | 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 + | colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.
Incubate at 50°C for 1 hour. - |- + - | colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes. + |} |}

## Revision as of 08:59, 8 December 2012

Project name Main project page
Previous entry      Next entry

## 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 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