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 Engineering PC-TFs
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Ligation/Assembly
- Weigh gel slices.
- Add three times amount of volume to each tube of gel (600 µL)
- Incubate at 55°C for 5-10 minutes.
- Centrifuge for a minute at 17,000 x g.
- Add 20 µL of wash buffer.
- Don't dump supernatent and centrifuge for one minute again.
- Place spin columns in 1.5 mL tubes.
Measure concentration of each fragment:
- Load Gen 5 2.0 software. Go to the Task Manager-Take 3 Application
- Go to Nucleic Acid Quantification.
- Pipette 2 µL of each DNA sequence into Take 3 plate.
- Place plate in EPOCH plate reader.
Results:
| Molarity of DNA Fragments
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| 1. KAH01 96.477 ng/µL
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| 2. KAH03 122.149 ng/µL
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| 3. KAH04 80.182 ng/µL
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| 4. KAH204 27.054 ng/µL
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| 5. KAH205 47.105 ng/µL
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| 6. KAH206 45.217 ng/µL
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| 7. KAH225 66.047 ng/µL
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- For all ligations, 25 ng of DNA vector = .3 µL
- There is a 2X molar ratio of insert:vector
| µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert)
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Assembly Scheme:
Number beside vector and insert is number of base pairs
- 1. hPCD (186) + KAH204 (2328)
- 2. hPCD (186) + KAH205 (1251)
- 3. hPCD (186) + KAH206 (1551)
- 4. hPCD (186) + KAH225 (1251)
- 5. hPCD (186) only
| '
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1
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2
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3
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4
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5
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| DNA Insert |
1.3 |
0.4 |
0.5 |
0.3 |
-----------
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| DNA Vector |
0.3 |
0.3 |
0.3 |
0.3 |
0.3
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| T4 Ligase |
1 |
1 |
1 |
1 |
1
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| Lign Buffer (2x) |
5 |
5 |
5 |
5 |
5
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| dH2O |
2.4 |
3.3 |
3.5 |
3.7 |
4
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10 µL |
10 µL |
10 µL |
10 µL |
10 µL
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- In a .5 mL tube, pipette the ligation buffer first.
- Then water, DNA insert, vector, and T4 ligase.
- Once completed, allow them to incubate for 10 minutes.
- Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes.
- Then follow transformation process:
Transformation Process
- Warm 100 μg/mL Amp agar plates at 37 °C
- Thaw fresh tube of DH5α Turbo cells on ice
- Add 30 μL of DH5α Turbo cells to DNA.
- Incubate cells + DNA on ice for 5 min.
- Label pre-warmed plates
- Transfer cells + DNA onto agar
- Add 10 - 15 sterile glass beads, shake, discard beads
- Incubate plates at 37 °C overnight
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Engineering PC-TFs
