04/29/15
- LCR - Phusion PCR for MV10
- KAH126 expression - rebuild PcTF expression plasmid for K562
MV10 Phusion PCR
- Use linearized MV10 (XbaI-cut)
Reagent
|
Rxn1
|
Rxn2
|
Expected: 1,2. MV10 = 5191
|
Hover name 10 μL/lane, 1% agarose; Ladder
|
Template |
0.5 |
0.5
|
10 uM fwd primer |
1.0 |
1.0
|
10 uM rev primer |
1.0 |
1.0
|
10 mM dNTPs |
1.0 |
1.0
|
Phusion pol. |
0.5 |
0.5
|
5x buffer |
10.0 HF |
10.0 GC
|
DMSO |
--- |
1.5
|
dH2O |
36.0 |
34.5
|
|
50.0 |
50.0
|
Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient
- 98°C, 3 min
- 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min]
- 72°C, 10 min
- 4°C ∞
Conclusion
Minipreps
Reagent
|
Volume
|
Expected: 1. BB 1 = size 2. BB2 = size
|
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
PstI |
1.0
|
dH2O |
9.5
|
|
15 μL --> 37°C/ ~15 min.
|
Assemblies
- BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
- BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
Reagent
|
Volume
|
|
DNA (plasmid) |
up to 25 μL
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~30 min.
|
Sample |
OD260 |
260/280 |
ng/μL
|
1. Digested part (a/b) |
--- |
--- |
---
|
2. Digested part (c/d) |
--- |
--- |
---
|
- Dephosphorylation (Roche)
Reagent
|
Volume
|
DNA (clean digest) |
up to 17 μL (500 ng)
|
10x buffer d.p. |
2.0
|
phosphatase |
1.0
|
dH2O |
---
|
|
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|
Ligation |
Plate results (lig : neg crtl) mm/dd/yy
|
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng |
new BioBrick #:1 (Pick #)
|
2. 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
|
|