01/24/11
- ✓ Minipreps: SP1AB w.t. (6), KAH160, 165, 170/pSB31 (6 each)
- ✓ Mutagenesis: site directed mutagenesis for SP1AB w.t.
- ✓ Cultures: KAH160 (10); 2 mL
Minipreps
> SP1AB w.t.: X/P
> Others: X/NcoI (BioBrick constructs contain 2 NcoI sites, vector has 1)
| Reagent |
Volume
|
Expected (approx.):
1-6. SP1AB w.t.
fwd = 3200, 580, 450, 300, 250
rev = 3650, 580, 300, 250
7-12. KAH160/pSB31
fwd = 636, 500+?
rev = 636, 500+?
13-18. KAH165/pSB31
fwd = 636, 344+?
rev = 636, 344+?
19-24. KAH170/pSB31
fwd = 444, 500+?
rev = 444, 500+?
Empty vector
XbaI = 4000, 650, 250, 200
NcoI should generate an extra band
|
15 μL/lane; 1% agarose
NcoI only
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| enzyme 1 |
1.0
|
| enzyme 2 |
1.0
|
| dH2O |
9.5
|
| |
15 μL --> 37°C/ ~15 min.
|
--> Conclusions: pSB31 does not have an NcoI site. NcoI should generate a short internal band in the BioBrick clones. Only KAH170 worked. Pick more colonies for KAH160 and 165 (10 each).
Site-directed Mutagenesis
> SP1AB has 3 PstI sites
> Stratagene Quick Change mutagenesis kit: Try all three primers at once
--> Forward primers: mut_SP1A 1, mut_SP1B 1, mut_SP1B 2
> Template is ~ 5kb
| 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/ ∞
(1/25/11)
> DpnI Digest (gets rid of methylated template DNA)
- Forward strand triple mutation
- 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
01/26/11
--> Results: Great ratio (one colony on neg ctrl.). Pick several and check by restriction digest.
|
Project name
