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 Karmella's BioBrick Cloning
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mm/dd/yy
- Assemblies: retry MV9
- T4 ligase quality check: Behzad's tube
Assemblies
- Try again, but with the old boil and cool method
- Oligo annealing
- See 03/19/13 for details about the oligos and reaction set-up
- Bring ~900 mL water to a boil in a large beaker (on a hot plate). Use a thermometer to check the temperature (>100°C).
- Float the oligo mixtures in the boiling water for 10 min. If there is condensation, carefully & quickly remove the tube and shoe the liquid down to the bottom and put the tubes back into the water.
- Turn off the heat source and allow the water bath to slowly cool to room temperature (25°C) for several hours.
- Digest (Fermentas FD) - MV2 vector, 03/20/13
- Measure conc. - 03/20/13
- Dephosphorylation (Roche) - MV2 XbaI-cut vector, 03/20/13
| Ligation |
Plate results (lig : neg crtl) 03/21/13
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| 1. A1 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng |
MV9 ##:##
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| 2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng |
MV9 ##:##
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| 3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng |
MV9 ##:##
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| 4. MV9(X dp)/ 25 ng |
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| 5. MV9(X no dp)/ 25 ng + Behzad's T4 ligase |
...
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| 6. MV9(X no dp)/ 25 ng |
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| |
1 |
2 |
3 |
4 |
9 |
10
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| Insert DNA |
0.5 |
1.0 |
2.0 |
--- |
--- |
---
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| Vector DNA |
1.0 |
1.0 |
1.0 |
1.0 |
0.3 |
0.3
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| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0
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| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
---
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| dH2O |
2.5 |
2.0 |
1.0 |
3.0 |
3.7 |
4.7
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| |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL
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- Pre-warm agar-amp plates with lids closed
- 30 min./ room temp
- Add 30 μL DH5α; 5 min/ ice
- Plate on 100 μg/mL amp; incubate at 37°C
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Karmella's BioBrick Cloning
