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Preparing another RBS-TesA and LacI Ligation
- Digested more of RBS-TesA with X and P(insert). Digested more of LacI with S and P (backbone).
- TesA+RBS insert with LacI+backbone
- Both samples used in the Clean and Concentrate kit
- Treat the LacI with Antarctic Phsophotase.
- Antarctic Phsophotase Treatment
| Reagent
|
RBS-TesA (μL)
|
LacI (μL)
|
| DNA |
15.0 |
15.0
|
| 10X fast Digest Buffer |
2.0 |
2.0
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| PstI |
1.0 |
1.0
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| XbaI |
1.0 |
.0
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| SpeI |
0.0 |
1.0
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| H2O |
1.0 |
1.0
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| 37°C for 1 hour
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- The RBS-TesA and LacI DNA samples were quantified:
- RBS-TesA: 39.157 ng/μL
- LacI: 62.579 ng/μL
- Overnight ligation reactions were conducted for the test ligation and the negative control.
- TesA+RBS insert with LacI+backbone
| Reagent
|
|
| LacI DNA |
10.0
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| Antarctic Phosphotase |
1.0
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| Phosphotase Buffer |
2.0
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| H2O |
7.0
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| 37°C for 1 hour then 65°C for 5 min
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| Reagent
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1 (μL)
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Control (μL)
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| TesA+RBS |
1.25 |
0.0
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| LacI |
1.0 |
1.0
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| Ligation Buffer |
1.0 |
1.0
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| Ligase |
1.0 |
1.0
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| H2O |
5.75 |
7.0
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| 16°C overnight
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