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 Building a Reporter Gene
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February 8, 2013
- Assembly: (5xGal4 : [Spacer : HSVtk TATA]) : (K : AmCyan : AmCyan : NLS : STOP : [polyA2]) (KAH201)/(KAH182); AmCyan reporter gene for chromatin proteins.
Assemblies
- Note, KAH201 is in vector V0120, so add 3200 bp to the size.
- KAH201/KAH182: (1) KAH201/(S/P)/203+3200 + (2) KAH182/(X/P)/1675
> Digests (Fermentas FD)
| Reagent |
Volume |
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30 μL/lane, 1% agarose; Ladder
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| DNA (plasmid) |
20 μL
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| 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 |
5.0 μL
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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. KAH201 (S/P) |
0.048 |
1.88 |
48.1
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| 2. KAH182 (X/P) |
0.008 |
1.93 |
8.1
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> Ligations
| Ligation |
Plate results (lig : neg crtl) 02/20/13
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| 1. KAH201 (S/P)/3403, 25 ng + KAH182 (X/P)/1675, # ng |
0
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| 2. KAH201 (X/P)/3403, 25 ng |
0
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1 |
2 |
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| Insert DNA |
0.74 |
--- |
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| Vector DNA |
0.52 |
0.52 |
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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 |
2.74 |
3.48 |
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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
- No colonies were formed, this will be repeated. See 2/26/13 entry.
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Building a Reporter Gene
