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 Engineering PC-TFs
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01/25/13
- Type IIS assembly - day 1
PCR
- Resuspend new primers to a final concentration of 100 μM
- Make 10 μM dilutions in separate tubes
- All of the reactions are as follows:
- pSB1A3, gg0001, gg0002
- template, primer 1, primer 2
- template, primer 1, primer 2
- template, primer 1, primer 2
| Reagent
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Volume
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Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL02 = 1431
4. BL03 = 1731
5. BL04 = 1686
6.fshPCD = 186
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10 μL/lane; 1% agarose; Ladder
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| DNA(plasmid) |
0.2 μL
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| primer 1 |
1.0
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| primer 2 |
1.0
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| 2x GoTaq |
25.0
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| dH2O |
22.8
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50.0 μL
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- 95°C, 3 min.
- [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
- 72°C, 3 min.
- 4°C, ∞
Purification
- Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
- Measure [DNA]
| Sample
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OD 260
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260/280
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ng/μL
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| 1. pSB1A3 |
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###
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| 2. ### |
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###
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| 3. ### |
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Engineering PC-TFs
