03/22/11
- ✓ Site directed mutagenesis: p56 and SP1AB mut2/3
Site-directed Mutagenesis
> Stratagene Quick Change mutagenesis kit.
- SP1AB (~5 kb): failed to mutate PstI site #1 last time because of incorrect primer. Ordered new primer mut_SP1A 1
- p65 (~4 kb): mutate w.t. clone (two PstI sites within single primer); mut_p65 1
| Reagent |
SP1AB mut2/3 |
p65 w.t.
|
| DNA (plasmid, ~100 ng)) |
1.0 |
0.5
|
| 10x buffer |
2.5 |
2.5
|
| Quick Solution |
0.5 |
0.5
|
| 10 μM primer 1 |
1.0 |
1.0
|
| dNTP mix |
1.0 |
1.0
|
| Quick Change enzyme mix |
1.0 |
1.0
|
| dH2O |
18.0 |
18.5
|
| |
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/ ∞
(3/23/11)
> DpnI Digest (gets rid of methylated template DNA)
- SP1AB mutation
- SP1AB control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
- p65 mutation
- 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 z-DH5α, use 10 μL each sample; Amp plates
03/24/11
--> Results
- SP1AB: 4 colonies (0 on neg. ctrl)
- p65: 2 colonies (0 on neg ctrl.)
|
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