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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
|
- Incubate at 37°C/ 10 min.
- Heat inactivate at 75°C/ 2 min.
- [final] = 200 ng/μL / 15 μL = ~13 ng/μL
| |
H2B |
LOV |
Negative Control |
H2B |
LOV |
Negative Control
|
| Insert DNA (2x mol vector) |
1.0 μL |
1.0 |
--- |
1.0 |
1.0 |
---
|
| Vector DNA (50 ng) |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5
|
| 2x Roche Rapid Ligation buffer |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0
|
| T4 ligase |
1.0 NEB |
1.0 NEB |
1.0 NEB |
1.0 Roche |
1.0 Roche |
1.0 Roche
|
| 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
|
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes.
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Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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