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 Karmella's BioBrick Cloning
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01/06/13
- Gibson Assembly: pick colonies & check via colony PCR
- Media: Make LB agar +amp
- Gibson Assembly: redo transformation with more DNA
Gibson Assembly results
- hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2)
- hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4)
- hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6)
- hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8)
- Pick and streak colonies from plates 3, 5, 7
- Use same pipette tip for colony PCR
| Reagent
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Volume
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5x mix
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30 μL/lane, 1% agarose; Ladder
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| Colony |
--- |
---
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| 10 μM primer BL9 |
1.0 |
5.0
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| 10 μM primer BL10 |
1.0 |
5.0
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| 2x GoTaq |
12.5 |
62.5
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| dH2O |
10.5 |
52.5
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25.0 μL |
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PCR
- 95°C, 3 min.
- [95°C, 30 sec.; 95°C, 30 sec.; 72°C, 1 min.] x35
- 72°C, 3 min.
- 4°C, ∞
Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C)
Transformation
- hPCD + BL01 + V0120, 6.0 μL 1/4x Gibson
- V0120, 6.0 μL 1/4x Gibson
- hPCD + BL01 + pSB1A3, 6.0 μL 1/4x Gibson
- pSB1A3, 6.0 μL 1/4x Gibson
- Add DNA to 50 μL DH5α-Turbo; ice/ 10 min.
- Plate on 100 μg/mL amp.
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Karmella's BioBrick Cloning
