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 Karmella's BioBrick Cloning
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01/10/13
- Violacein: successful growth; re-streak
- Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13
- Golden gate trial 2: new PCR primers
Violacein
- Plate 1: Kan plasmid
- Plate 2: Amp plasmid (violacein exp.)
Golden gate trial 1
- Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
Golden gate trial 2
- Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region
| Reagent
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Volume
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Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520
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| DNA(plasmid) |
0.2 μL
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| primer 1 |
1.0
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| primer 2 |
1.0
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| 2x GoTaq |
25.0
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| dH2O |
22.8
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50.0 μL
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PCR
- 95°C, 3 min.
- [95°C, 3 min.; 95°C, 3 min.; 95°C, 3 min.] x35
- 72°C, 3 min.
- 4°C, ∞
| Sample
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OD 260
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260/280
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ng/μL
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| 1. pSB1A3 |
0.126 |
1.888 |
77.357
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| 2. hPCD |
0.069 |
2.192 |
17.025
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| 3. BL01 |
0.121 |
1.898 |
66.878
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Karmella's BioBrick Cloning
