12/22/09
- ✓ Minipreps: KAH93-95/MV2 (2 each)
- ✓ Transfection 1: KAH93, 94, 95/MV2 for microscopy and RT-PCR (6-well, Lipo)
- ✓ Transfection 2: KAH94, 95/MV2 for miRNA dose response curve (24-well, Fugene)
- ✓ Re-transform plasmids: KAH93, 94, 95/MV2 (Did not work well, re-try with Z-DH5α, 12/23/09 ✓)
Minipreps
> Check with E/P digests
Reagent |
Volume
|
Expected: 1,2. KAH93/MV2 = 4839 3,4. KAH94/MV2 = 5011 3,4. KAH95/MV2 = 5083
|
15 μL/lane; 1% agarose
|
DNA(plasmid) |
2.0
|
10X buffer |
1.5
|
EcoRI |
1.0
|
PstI |
1.0
|
dH2O |
9.5
|
|
15 μL --> 37°C/ ~15 min.
|
> No plasmid DNA. Re-transform from minipreps (quick & dirty transformation, DH5α T1R lab stock)
Transfection 1
> Details
Wells |
Sample |
DNA |
Volume |
Lipo |
Opti-MEM (total)
|
1 |
KAH93/MV2 |
2 μg |
4.4 μL |
4 μL |
500 μL
|
2 |
KAH93/MV2(Dox) |
" |
4.4 μL |
" |
"
|
3 |
KAH94/MV2 |
" |
4.8 μL |
" |
"
|
4 |
KAH94/MV2(Dox) |
" |
4.8 μL |
" |
"
|
5 |
KAH95/MV2 |
" |
8.7 μL |
" |
"
|
6 |
KAH95/MV2(Dox) |
" |
8.7 μL |
" |
"
|
7 |
KAH108/MV2 |
" |
4.6 μL |
" |
"
|
8 |
KAH108/MV2(Dox) |
" |
4.6 μL |
" |
"
|
9 |
mock |
--- |
--- |
" |
"
|
10 |
mock(Dox) |
--- |
--- |
" |
"
|
11 |
untreated |
--- |
--- |
--- |
"
|
12 |
untreated(Dox) |
--- |
--- |
--- |
"
|
> Add 2x Lipo to 500 μL Opti-MEM --> R.T/ 5 min.
> Add 2x DNA to 500 μL Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Remove 500 μL medium from each well; Add 500 μL complexes to each well (4 ml total vol.); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
Day 2
> Add 1μg/mL Dox to Dox+ samples. Incubate overnight (~20 hours).
Day 3
> RNA preps/ cDNA synthesis
Transfection 2
> Details
Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
1-6 |
KAH94/MV2 |
150 ng |
0.4 μL |
1.8 μL |
18.2 μL
|
7-12 |
KAH95/MV2 |
" |
1.2 μL |
" |
"
|
13-18 |
KAH108/MV2(ctrl) |
" |
0.3 μL |
" |
"
|
19-24 |
mock |
" |
--- |
" |
"
|
> Master mixes (4, x6): 10.8 μL Fugene + 109.2 μL Opti-MEM --> R.T/ 5 min.
> Add 6x vol DNA --> R.T./ 20 min.
> Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37°C overnight
Day 2
> Refresh w/ ab-free, no phenol red medium
> Add varying doses of doxycycline to the wells in each row: 0, 0.1, 1.0, 10.0, 100.0, 1000.0 ng/mL
---> 1:10 serial dilutions; dilute dox in 50 μL medium
> Assay RFP/YFP after 5 or 6 hours (plate reader)
--> Accidentally did 1:100 serial dilution (0, 0.00001, 0.001, 0.1, 10.0, 1000.0) for Read #1
--> Refreshed medium and used correct 1:10 dilutions of Dox for overnight induction
> Grow overnight
Day 3
> Read #2: Assay RFP/YFP (morning)
|