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 Karmella's BioBrick Cloning
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04/12/13
- Chem. Competent cell prep: CaCl wash; 15% glycerol/CaCl resuspension (used 30 mL instead of 20 mL); incubate in cold room on ice o/n
- MV9 building: colony PCR
MV9 building: colony PCR
- Pick 8 colonies from plates 1, 2, 3, and 4; make a mini-streak library, then wash pipette tip in PCR master mix
- Pick 4 colonies from plate 5 (neg. ctrl)
- Primers for MV2 vector: DD122B (fwd), DD123B (rev); see BBa_J176122
| Reagent
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Volume
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x38
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Expected:
MV2 (empty) = 177
MV9 (single insert) = 253
extra inserts = +76
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10 μL/lane, 1% agarose; Ladder
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| DNA (colony) |
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---
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| 10 μM primer 1 |
1.0 |
38.0
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| 10 μM primer 2 |
1.0 |
38.0
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| 2x GoTaq green |
10.0 |
380.0
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| dH2O |
8.0 |
304.0
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20 μL |
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Thermal cycling
- 95°C, 3 min.
- [95°C, 15 sec; 57°C, 15 sec; 72°C, 15 sec] x30
- 72°C, 3 min.
- 4°C, ∞
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Karmella's BioBrick Cloning
