04/20/11
- ✓ qRT-PCR: Pc-TF plates 27, 28
- ✓ cDNA: HEK-Gal4-luc Pc-TF RNA preps 1-8 (no silencing), 9-15 (2-day dox set, remainder), 17-24 (4-day dox set)
- ✓ qRT-PCR: HEK-Gal4-luc Pc-TF cDNA plate HEK1
qRT-PCR
> Plate 27
--> 750 nM Primers (& cDNA dilution)
- IGFBP2 51A (1:10)
--> Templates, use 2 μL
- KAH126-1 (2/21/10)
- KAH126-1 +dox "
- KAH126-1 (6/15/10)
- KAH126-1 +dox "
- KAH126-1 (7/26/10)
- (no template)
- KAH128-8 (2/21/10)
- (no template)
- KAH128-8.3 (7/26/10)
- KAH128-8.3 +dox "
- KAH129-4 (2/21/10)
- KAH129-4 +dox "
- KAH129-4 (7/26/10)
- KAH129-4 +dox "
- KAH154-2 (6/15/10)
- KAH154-2 +dox "
- KAH154-2 (7/26/10)
- (no template)
- KAH154-2 (4/08/11 B)
- KAH154-2 +dox "
- KAH156-5 (7/26/10)
- KAH156-5 +dox "
- KAH156-5 (4/08/11 A)
- KAH156-5 +dox "
- KAH156-5 (4/08/11 B)
- KAH156-5 +dox "
- KAH157-1 (7/26/10)
- KAH157-1 +dox "
- KAH157-1 (4/08/11 A)
- KAH157-1 +dox "
- KAH157-1 (4/08/11 B)
- KAH157-1 +dox "
> Plate 25
--> 750 nM Primers (& cDNA dilution)
- IGFBP2 51A (1:10)
--> Templates, use 2 μL
- KAH132-8 (6/15/10)
- KAH132-8 +dox "
- KAH132-8 (6/18/10)
- KAH132-8 +dox "
- KAH132-8 (6/23/10)
- KAH132-8 +dox "
- FTRx (2/21/10)
- FTRx +dox "
- FTRx (6/15/10)
- FTRx +dox "
- FTRx (6/18/10)
- FTRx +dox "
- FTRx (7/26/10)
- FTRx +dox "
Reagent |
1 rxn |
Primer mix (x49), make 2 |
Primer mix (x43)
|
diluted cDNA |
2.0 |
--- |
---
|
SYBR Green mix |
7.5 |
367.5 |
322.5
|
750 nM primers |
3.0 |
147.0 |
129.0
|
dH2O |
2.5 |
122.5 |
107.5
|
|
15.0
|
--> Aliquot 39.0 primer mix into 1st well of each 3x set
--> Add 6.0 (2.0 x3) DNA to 39.0 primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in each 3x set
Bio-Rad CFX96 qPCR (Kirschner lab)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film
- 95°C/ 5 min.
- [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x45
- Melt curve range 58°C -> 95°C/ 0.5°C per step
cDNA synthesis
--> Samples: Cells treated with dox for 0, 2, 4 days; transfected with Pc-TF/MV1 constructs 5 days after dox wash-out
Sample |
A260 |
260/280 |
ng/ul
|
1. 0 dox/ 160 |
1.461 |
1.74 |
58.4
|
2. 0 dox/ 165 |
1.215 |
1.56 |
48.6
|
3. 0 dox/ 161 |
0.964 |
1.64 |
38.6
|
4. 0 dox/ 167 |
0.995 |
1.61 |
39.8
|
5. 0 dox/ 162 |
1.677 |
1.62 |
67.1
|
6. 0 dox/ 166 |
1.159 |
1.67 |
46.3
|
7. 0 dox/ 170 |
1.283 |
1.76 |
51.3
|
8. 0 dox/ mock |
1.359 |
1.77 |
54.4
|
9. 2dy dox/ 160 |
1.662 |
1.62 |
66.5
|
10. 2dy dox/ 165 |
1.282 |
1.54 |
51.3
|
11. 2dy dox/ 161 |
1.289 |
1.56 |
51.6
|
12. 2dy dox/ 167 |
1.387 |
1.67 |
55.5
|
13. 2dy dox/ 162 |
1.157 |
1.64 |
46.3
|
14. 2dy dox/ 166 |
1.156 |
1.70 |
46.2
|
15. 2dy dox/ 170 |
3.624 |
1.89 |
145.0
|
16. done
|
17. 4dy dox/ 160 |
4.388 |
1.90 |
175.5
|
18. 4dy dox/ 165 |
4.293 |
1.89 |
171.7
|
19. 4dy dox/ 161 |
4.907 |
1.87 |
196.3
|
20. 4dy dox/ 167 |
4.156 |
1.88 |
166.3
|
21. 4dy dox/ 162 |
3.799 |
1.90 |
152.0
|
22. 4dy dox/ 166 |
4.175 |
1.90 |
167.0
|
23. 4dy dox/ 170 |
4.421 |
1.90 |
176.8
|
24. 4dy dox/ mock |
4.987 |
1.92 |
199.5
|
> oligo(dT) Primer annealing
--> Do 4 rxn's for 129-4 #1
--> 2 rxn's ea. for 129-4 +dox #4 & #5
Reagent |
Vol
|
total RNA (up to 2μg) |
8.0
|
50μM oligo(dT) primer |
1.0
|
10 mM dNTP mix |
1.0
|
DEPC-treated water |
---
|
|
10.0 μL --> 65°C/ 5 min.; ice/ 1 min.
|
> cDNA synthesis mix
--> 16 reactions total
Reagent |
Vol |
Mix (x23)
|
10x RT buffer |
2.0 |
46.0
|
25 mM MgCl2 |
4.0 |
92.0
|
0.1 M DDT |
2.0 |
46.0
|
RNaseOUT |
1.0 |
23.0
|
SuperScript III RT |
1.0 |
23.0
|
|
10.0 μL |
|
--> Add 10 μL mix to each annealing rxn.
--> 50°C/ 50 min., 80°C/ 5 min., ice
--> Add 0.8 μL RNase H, 37°C/ 20 min.
--> Store at -20°C
qRT-PCR
> Plate HEK1
--> 750 nM Primers (& cDNA dilution)
- GAPDH 21A (1:100)
- mCh1 "
- luc1 "
- luc4 "
--> Templates, use 2 μL
- (17) 4dy dox/ 160
- (18) 4dy dox/ 165
- (19) 4dy dox/ 161
- (20) 4dy dox/ 167
- (21) 4dy dox/ 162
- (22) 4dy dox/ 166
- (23) 4dy dox/ 170
- (24) 4dy dox/ mock
Reagent |
1 rxn |
Primer mix (x25)
|
diluted cDNA |
2.0 |
---
|
SYBR Green mix |
7.5 |
187.5
|
750 nM primers |
3.0 |
75.0
|
dH2O |
2.5 |
62.5
|
|
15.0
|
--> Aliquot 39.0 primer mix into 1st well of each 3x set
--> Add 6.0 (2.0 x3) DNA to 39.0 primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in each 3x set
Bio-Rad CFX96 qPCR (Kirschner lab)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film
- 95°C/ 5 min.
- [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x45
- Melt curve range 58°C -> 95°C/ 0.5°C per step
|