07/07/11
- ✓ Minipreps: mCherry, 5xGal4 retransformations
- ✓ Assemblies: KAH204, 205, 206, 207, 208, 209
- ✓ Site directed mutagenesis: p65 (new primers)
- ✓ Digest: investigate weird miR-luc sensor constructs; check KAH196, 177, 198/MV8
Minipreps
> Check with E/P digests
Reagent |
Volume
|
Expected: 1. mCherry = 705 2. 5xGal4 = 93
|
15 μL/lane; 1% agarose
|
DNA(plasmid) |
3.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
PstI |
1.0
|
dH2O |
8.5
|
|
15 μL --> 37°C/ ~15 min.
|
Assemblies
- KAH204: (1) mCh/(S/P)/705 + (2) SP1AB/(X/P)/1617
- KAH205: " + (3) SP1A/(X/P)/540
- KAH206: " + (4) SP1B/(X/P)/840
- KAH207: (5) KAH203/(S/P)/155 + (8) KAH182/(X/P)/1675
- KAH208: (6) KAH201/(S/P)/203 + "
- KAH209: (7) KAH202/(S/P)/251 + "
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
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.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. mCh (S/P) |
0.972 |
1.86 |
48.6
|
2. SP1AB (X/P) |
0.170 |
6.31 |
8.5
|
3. SP1A (X/P) |
0.268 |
2.50 |
13.4
|
4. SP1B (X/P) |
0.298 |
2.46 |
14.9
|
5. KAH203 (S/P) |
0.153 |
1.89 |
7.7
|
6. KAH201 (S/P) |
0.128 |
2.53 |
6.4
|
7. KAH202 (S/P) |
0.118 |
7.22 |
5.9
|
8. KAH182 (X/P) |
0.120 |
3.76 |
6.0
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 07/08/11
|
1. SP1AB(X/P)/1617, 17 ng + mCh(S/P)/~3905, 20 ng |
KAH204 >10:1 (Pick 2)
|
2. SP1A(X/P)/540, 6 ng + " |
KAH205 >10:1 (Pick 2)
|
3. SP1B(X/P)/840, 9 ng + " |
KAH206 >10:1 (Pick 2)
|
4. mCh(S/P)/ 20 ng |
|
5. KAH182(X/P)/1675, 20 ng + KAH203(S/P)/~3355, 20 ng |
KAH207 >10:1 (Pick 2)
|
6. KAH203(S/P)/ 20 ng |
|
7. KAH182(X/P)/1675, 20 ng + KAH201(S/P)/~3403, 20 ng |
KAH208 >10:1 (Pick 2)
|
8. KAH201(S/P)/ 20 ng |
|
9. KAH182(X/P)/1675, 19 ng + KAH202(S/P)/~3451, 20 ng |
KAH209 >10:1 (Pick 2)
|
10. KAH201(S/P)/ 20 ng |
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10
|
Insert DNA |
2.5 |
0.5 |
1.0 |
--- |
3.0 |
--- |
3.0 |
--- |
3.0 |
---
|
Vector DNA |
0.5 |
0.5 |
0.5 |
0.5 |
2.5 |
2.5 |
3.0 |
3.0 |
3.0 |
3.0
|
2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
6.5 |
6.5 |
7.0 |
7.0 |
7.0 |
7.0
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
dH2O |
1.0 |
3.0 |
2.5 |
3.5 |
--- |
3.0 |
--- |
3.0 |
--- |
3.0
|
|
10 μL |
10 μL |
10 μL |
10 μL |
13 μL |
13 μL |
14 μL |
14 μL |
14 μL |
14 μL
|
Site-directed Mutagenesis
> Stratagene Quick Change mutagenesis kit.
- p65 (~4 kb): tried to mutate w.t. clone (two PstI sites within single primer); last attempt mutated only the first site (3/25/11)
--> Try to mutate second PstI site with forward and reverse primers...
- Primer mut_p65 2 (plus strand)
- Primer mut_p65 2 (minus strand)
Reagent |
p65 w.t.
|
DNA (plasmid, ~100 ng) |
1.0
|
10x buffer |
2.5
|
Quick Solution |
0.5
|
10 μM primer 1 |
1.0
|
dNTP mix |
1.0
|
Quick Change enzyme mix |
1.0
|
dH2O |
18.0
|
|
25 μL
|
--> BioRad PCR (Block A)
- 95°C/ 1 min.
- [95°C/ 1 min., 55°C/ 1 min., 65°C/ 8 min (2 min./kb)] x30
- 65°C/ 1 min.
- 4°C/ ∞
(7/08/11)
> DpnI Digest (gets rid of methylated template DNA)
- p65 + strand mutation
- p65 - strand 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
07/09/11
--> Results
- p65 +strand mut: 0 colonies (0 on neg ctrl.)
- p65 -strand mut: 0 colonies (0 on neg ctrl.)
Minipreps
> Check with ApaLI
> Refer to 6/04/11 for correct 196, 198 bands (E/ApaLI)
Reagent |
Volume
|
Expected: MV8 bands = 1243, 1135, 288 1. KAH196/MV8 (6/05/11) = **2772, *1668, 711 2. KAH177/MV8 (1/020/11) = **2772, *1835, 711 3. KAH198/MV8 (6/05/11) = **2772, *1915, 711 *first frag. + 234 **last frag + 1655
|
15 μL/lane; 1% agarose
|
DNA(plasmid) |
3.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
PstI |
1.0
|
dH2O |
8.5
|
|
15 μL --> 37°C/ ~15 min.
|
--> Everything looks correct. Use these minipreps for re-cloning final miR-luc sensor set.
--> Note: pre-cut vector KAH177/MV8 worked for 183-177/MV8 (see 6/07/11); continue using this vector)
--> Re-cut 196 and 198/MV8, throw out old cut vectors
|