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Karmella's BioBrick Cloning
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11/30/12
- Assembly: CMVTetO2:K (KAH3)/ MV; expression vector for Vi's mammalian cell expression experiment
Assemblies
- KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(X/P)/4529
> Digests (Fermentas FD)
Reagent |
Volume |
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30 μL/lane, 1% agarose; Ladder
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DNA (plasmid) |
up to 25 μL
|
10x buffer |
3.0
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enzyme 1 |
1.0
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enzyme 2 |
1.0
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dH2O |
---
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30 μL --> 37°C/ ~15 min.
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> Measure conc.'s of gel purified fragments
Sample |
OD260 |
260/280 |
ng/μL
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1. KAH3 (X/P) |
0.028 |
1.96 |
28.41
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2. MV2 (S/P) |
0.085 |
1.93 |
85.02
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> Ligations
Ligation |
Plate results (lig : neg crtl) 11/31/12
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1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng |
KAH3/MV2 >10:1 (Pick 2)
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2. MV2 (S/P)/ 25 ng |
zero colonies
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|
1 |
2 |
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Insert DNA |
0.3 |
--- |
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Vector DNA |
0.3 |
0.3 |
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2x lgn buf (Roche) |
5.0 |
5.0 |
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T4 ligase (NEB) |
1.0 |
1.0 |
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dH2O |
3.4 |
3.7 |
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10 μL |
10 μL |
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- Follow quick transformation protocol
- Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
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