04/11/13
- Oligo assembly for new mammalian vector MV9
- Chem. Competent cell prep: DH5α Turbo (from NEB); 5 mL culture/ ~8 hours; 2x 250 mL cultures o/n
Assemblies
- Try again with the old boil and cool method...
- Aluminum foil lid on the beaker to prevent condensation
- Different amounts of 100 μM oligos
- 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. Cover the beaker with aluminum foil to keep the air above the tube warm.
- Turn off the heat source and allow the aluminum foil-covered water bath to slowly cool to room temperature (25°C) for several hours.
| |
1 |
2 |
3 |
4
|
| 100 μM Oligo 1 |
3.0 |
2.0 |
1.0 |
0.5
|
| 100 μM Oligo 2 |
3.0 |
2.0 |
1.0 |
0.5
|
| 100 μM Oligo 3 |
3.0 |
2.0 |
1.0 |
0.5
|
| 100 μM Oligo 4 |
3.0 |
2.0 |
1.0 |
0.5
|
| 10x annealing buffer |
2.0 |
2.0 |
2.0 |
2.0
|
| dH2O |
6.0 |
10.0 |
14.0 |
16.0
|
| |
20 μL |
20 μL |
20 μL |
20 μL
|
- Digest (Fermentas FD) - MV2 vector, 03/20/13
- Measure conc. - 03/20/13
- Dephosphorylation (Roche) - MV2 XbaI-cut vector, 03/20/13
- Ligations
- Inserts have no 5'-phosphates, so dp vector will not work! Switch to non-dp vector. expect high bg on neg ctrl. Will need to screen ligations.
| Ligation |
Plate results (lig : neg crtl) 03/22/13
|
| 1. 1 (ds oligo S/S)/76, 0.5 μL + MV2(X)/4529, 25 ng |
20 colonies
|
| 2. 2 (ds oligo S/S)/76, 0.5 μL + MV2(X)/4529, 25 ng |
20 colonies
|
| 3. 3 (ds oligo S/S)/76, 0.5 μL + MV2(X)/4529, 25 ng |
20 colonies
|
| 4. 3 (ds oligo S/S)/76, 0.5 μL + MV2(X)/4529, 25 ng |
20 colonies
|
| 5. MV2(X dp)/ 25 ng |
~100 colonies
|
| |
1 |
2 |
3 |
4 |
5
|
| Insert DNA |
0.5 |
0.5 |
0.5 |
0.5 |
---
|
| Vector DNA |
0.3 |
0.3 |
0.3 |
0.3 |
0.3
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
5.0
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
| dH2O |
3.2 |
3.2 |
3.2 |
3.2 |
3.7
|
| |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL
|
- Incubate ligation for 30 min./ room temp
- Pre-warm agar-amp plates with lids closed
- Add 30 μL DH5α (fresh from NEB); 2 min/ ice
- Heat shock at 42°C, 30 sec. Place immediately on ice.
- Add 100 μL plain LB to cells.
- Plate on 100 μg/mL amp; incubate at 37°C
4/12/13
- More colonies on the neg. ctrl. than the insert + vector plates.
- Do colony PCR using MV2 primers; pick 8 colonies from each plate 1 - 4; also do PCR on 4 colonies from the neg. ctrl.
|
Karmella's BioBrick Cloning
