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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
- KAH201/KAH182: (1) KAH201/(X/P)/203 + (2) KAH182/(S/P)/1675
> Digests (Fermentas FD)
Reagent |
Volume |
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30 μL/lane, 1% agarose; Ladder
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DNA (plasmid) |
15 to 25 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 |
0 to 10 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 (X/P) |
TBD |
TBD |
TBD
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2. KAH182 (S/P) |
TBD |
TBD |
TBD
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> Ligations (edit after ligation is done)
Ligation |
Plate results (lig : neg crtl) 11/31/12
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1. KAH201 (X/P)/203, x ng + KAH182 (S/P)/1675, x ng |
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2. KAH182 (S/P)/ 25 ng |
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1 |
2 |
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Insert DNA |
--- |
--- |
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Vector DNA |
--- |
--- |
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2x lgn buf (Roche) |
--- |
--- |
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T4 ligase (NEB) |
--- |
--- |
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dH2O |
--- |
--- |
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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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