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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
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- 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
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| 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
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| LOV-H2B LOV insert DNA (1x mol vector) |
0 μL |
1.0 |
0
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| Vector DNA (39 ng) |
3.0 |
3.0 |
3.0
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| Gibson master mix |
15.0 |
15.0 |
15.0
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Mix the reaction(s) thoroughly by flicking the tube.
Incubate at 50°C for 1 hour.
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Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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