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
| Volume
| 
30 μL/lane, 1% agarose; Ladder
|
| DNA (plasmid) | 20.0 μL
|
| 10x buffer | 3.0
|
| XbaI | 2.0
|
| dH2O | 5.0
|
| | 30 μL --> 37°C/ ~15 min.
|
- Gel purify using the Zymo DNA Gel Recovery kit.
- Elute with 20 μL dH2O
| Sample | OD260 | 260/280 | ng/μL
|
| 1. MV2 (XbaI) | 0.081 | 1.841 | 81.3
|
- Dephosphorylation (Roche)
| Reagent
| Volume
|
| MV2 (XbaI) | 6.2 μL (500 ng)
|
| 10x buffer d.p. | 2.0
|
| rapid alk. phosphatase | 1.0
|
| dH2O | 10.8
|
| | 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|
| Ligation | Plate results (lig : neg crtl) 03/21/13
|
| 1. A1 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng | MV9 failed
|
| 2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng | MV9 failed
|
| 3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng | MV9 failed
|
| 4. MV9(X dp)/ 25 ng |
|
| 5. A3 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng | MV9 failed
|
| 6. A3 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng | MV9 failed
|
| 7. A3 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng | MV9 failed
|
| 8. MV9(X dp)/ 25 ng |
|
| 9. MV9(X no dp)/ 25 ng + T4 ligase | Re-ligation successful, ~40 colonies
|
| 10. MV9(X no dp)/ 25 ng |
|
| | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10
|
| 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 | 0.3 | 0.3
|
| 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 | ---
|
| dH2O | 2.5 | 2.0 | 1.0 | 3.0 | 2.5 | 2.0 | 1.0 | 3.0 | 3.7 | 4.7
|
| | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL
|
- 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
|
Karmella's BioBrick Cloning
Main project page
Previous entry Next entry
