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
| 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
|
Project name
Main project page
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