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. This way, the vector can be customized to carry different promoters.
- Oligo annealing
- 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
|
|
DNA (plasmid) |
20.0 μL
|
10x buffer |
3.0
|
XbaI |
2.0
|
dH2O |
5.0
|
|
30 μL --> 37°C/ ~15 min.
|
Sample |
OD260 |
260/280 |
ng/μL
|
1. MV2 (XbaI) |
0.081 |
1.841 |
81.3
|
- Dephosphorylation (Roche)
Reagent
|
Volume
|
DNA (clean digest) |
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. insert(Oligo S/S)/size, ## ng + MV2(X dp)/size, 25 ng |
MV9 #:1 (Pick #)
|
2. insert(Oligo S/S)/size, ## ng + vector(c/d)/size, ## ng |
new BioBrick #:1 (Pick #)
|
3. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng |
new BioBrick #:1 (Pick #)
|
4. vector(c/d)/ ## ng |
|
|
1 |
2 |
|
Insert DNA |
### |
--- |
|
Vector DNA |
### |
### |
|
2x lgn buf (Roche) |
### |
### |
|
T4 ligase (NEB) |
1.0 |
1.0 |
|
dH2O |
### |
### |
|
|
# μL |
# μL |
|
Oligo annealing
- New BB 1
- New BB 2
DNA (oligos, 100 μM) |
up to 18 μL (3 μL ea.)
|
10x annealing buffer |
2.0
|
dH2O |
---
|
|
20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|
|