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 Engineering PC-TFs
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2/9/2013
- 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
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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
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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 2-09-2013)
- Clean with Zymo DNA clean &concentrator kit; eluted w/ 20 μL dH2O
- Measure [DNA] (1-29-2013)
| 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 |
.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
