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| | ==Summary== | | ==Summary== |
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| - | * Type IIS assembly - day 1 | + | * Type IIS assembly - PCR products |
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Revision as of 19:27, 14 February 2013
 Engineering PC-TFs
|  Main project page
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Summary
- Type IIS assembly - PCR products
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
- hPCD, gg0003, gg0004
- BL02, gg0005, gg0007
- BL03, gg0005, gg0008
- BL04, gg0005, gg0009
| Reagent
| Volume
| 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 (Completed on 1-25-2013)
- Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
- Measure [DNA] (1-29-2013)
| Sample
| OD 260
| 260/280
| ng/μL
|
| 1. pSB1A3 | .079 | 1.837 | 79.379
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| 2. hPCD | .022 | 1.883 | 22.239
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| 3. BL02 | .129 | 1.883 | 128.889
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| 4. BL03 | .079 | 1.918 | 78.72
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| 5. BL04 | .06 | 1.879 | 59.912
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Engineering PC-TFs
