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 Karmella's BioBrick Cloning
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03/20/13
- Oligo annealing, round 2
- Assembly: annealed part into MV2
Assemblies
- New strategy for mammalian vector:
- Insert K-XbaI-NLS-6his-stop into the puro vector that has a deleted promoter (MV2, puro). This way, the vector can be customized to carry different promoters.
- Insertion of the ds oligo should destroy the vector's XbaI site
- Will need to check inserts for copy number and orientation
- Oligo annealing
- See 03/19/13 for details about the oligos and reaction set-up
- Redo oligo annealing with 99.9 and 97.9°C starting temperatures...
- Previous Thermal cycling (3/19/13)
- 99.9, 10 min.
- [99.9, 1 min., -2°C; 90.0, 1 min., -2°C] x35
- 25°C, ∞
- Labeled tubes A1, A2; Stored on bench o/n
- New Thermal cycling (3/20/13)
- 99.9, 10 min.
- [99.9, 1 min., -2°C; 97.9, 1 min., -2°C] x36
- 25°C, ∞
- Labeled tubes A3, A4
| Reagent
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Volume
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30 μL/lane, 1% agarose; Ladder
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| DNA (plasmid) |
20.0 μL
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| 10x buffer |
3.0
|
| XbaI |
2.0
|
| dH2O |
5.0
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| |
30 μL --> 37°C/ ~15 min.
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- Gel purify using the Zymo DNA Gel Recovery kit.
- Elute with 20 μL dH2O
| Sample |
OD260 |
260/280 |
ng/μL
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| 1. MV2 (XbaI) |
0.081 |
1.841 |
81.3
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- Dephosphorylation (Roche)
| Reagent
|
Volume
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| MV2 (XbaI) |
6.2 μL (500 ng)
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| 10x buffer d.p. |
2.0
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| rapid alk. phosphatase |
1.0
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| dH2O |
10.8
|
| |
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
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| 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 failed
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| 2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng |
MV9 failed
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| 3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng |
MV9 failed
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| 4. MV9(X dp)/ 25 ng |
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| 5. A3 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng |
MV9 failed
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| 6. A3 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng |
MV9 failed
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| 7. A3 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng |
MV9 failed
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| 8. MV9(X dp)/ 25 ng |
|
| 9. MV9(X no dp)/ 25 ng + T4 ligase |
Re-ligation successful, ~40 colonies
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| 10. MV9(X no dp)/ 25 ng |
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10
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| Insert DNA |
0.5 |
1.0 |
2.0 |
--- |
0.5 |
1.0 |
2.0 |
--- |
--- |
---
|
| Vector DNA |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
---
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| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
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| dH2O |
2.5 |
2.0 |
1.0 |
3.0 |
2.5 |
2.0 |
1.0 |
3.0 |
3.0 |
4.0
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| |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL
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- 10 min./ room temp
- Add 30 μL DH5α; 5 min/ ice
- Plate on 100 μg/mL amp; incubate at 37°C
3/21/13
Results for plates 9 and 10
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Karmella's BioBrick Cloning
