02/23/11
- ✓ Cultures/ minipreps: G luc (4); 2 mL each (sequence confirm tomorrow)
- Mutagenesis: activation domains SP1AB 2/3 (mutate PstI site #1); p65 (wait for new primer)
--> mut_SP1A 1 is incorrect; re-order
Minipreps
> Check with E/P digests
| Reagent |
Volume
|
Expected:
1-4. G luc (KAH016) = 558
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| PstI |
1.0
|
| dH2O |
9.5
|
| |
15 μL --> 37°C/ ~15 min.
|
Site-directed Mutagenesis
> SP1AB: Mutated PstI sites 2 and 3 last time; still need to mutate site #1
> Stratagene Quick Change mutagenesis kit:
- SP1AB 2/3, primer mut_SP1A 1
- p65, primer mut_p65 1
> Templates are ~ 5kb and 4kb
| Reagent |
Volume |
|
| DNA (plasmid) |
1.0 (~100 ng)
|
| 10x buffer |
2.5
|
| Quick Solution |
0.5
|
| 10 μM primer 1 |
1.0
|
| 10 μM primer 2 |
1.0
|
| 10 μM primer 3 |
1.0
|
| dNTP mix |
1.0
|
| Quick Change enzyme mix |
1.0
|
| dH2O |
16.0
|
| |
25 μL
|
--> BioRad PCR (Block A)
- 95°C/ 1 min.
- [95°C/ 1 min., 55°C/ 1 min., 65°C/ 10 min (2 min./kb)] x30
- 65°C/ 1 min.
- 4°C/ ∞
(2/24/11)
> DpnI Digest (gets rid of methylated template DNA)
- SP1AB 2/3 mut rxn
- SP1AB 2/3 control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
- p65 mut rxn
- p65 control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
--> Add 1 μL DpnI enzyme to each sample
--> 37°C/ 1 hr.
--> Transform 30 μL DH5α Turbo, use 10 μL each sample; Amp plates
2/25/11
--> Results
|
Project name
Main project page
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