01/04/10
- RT-PCR: RFP reporter-transfected samples + controls
RT-PCR
> Round 1: Controls KAH108 (RFP), KAH108 +Dox (RFP, YFP), mock (untransfected)
--> Use dilutions of cDNA: 1. 1:10, 2. 1:100, 3. 1:1000, 4. 1:10000
--> 12 templates x 6 primer pairs YFP1, YFP2, RFP1, RFP2, MV2 (vector backbone), GAPDH
--> Make 13x master mix for each primer pair
| Reagent |
Volume |
x13 |
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Expected sizes = 100 bp
1-4. KAH108/YFP1
5-8. KAH108/YFP2
9-12. KAH108/RFP1
13-16. KAH108/RFP2
19-20. KAH108/MV2
21-24. KAH108/GAPDH
25-28. KAH108+Dox/YFP1
29-32. KAH108+Dox/YFP2
33-36. KAH108+Dox/RFP1
37-40.KAH108+Dox/RFP2
41-44. KAH108+Dox/MV2
45-48. KAH108+Dox/GAPDH
49-52. mock/YFP1
53-56. mock/YFP2
57-60. mock/RFP1
61-64. mock/RFP2
65-68. mock/MV2
69-72. mock/GAPDH
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10 μL/lane, 1% agarose
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| cDNA |
0.5 |
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| 10μM primer mix |
2.0 |
26.0
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| 2x GoTaq Green |
10.0 |
130.0
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| dH2O |
7.5 |
97.5
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20.0 |
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--> PCR: 95°C/ 3 min.; [95°C/ 30 sec, 57°C/ 30 sec, 72°C/ 30 sec] x 30; 72°C/ 3 min.; 4°C/ ∞
> Round 2: All samples
--> Use 1:1000 dilution of cDNA
--> 10 templates x 4 primer pairs: 1. YFP2, 2. RFP1, 3. GAPDH, 4. MV2 (vector backbone)
--> Make 11x master mix for each primer pair
| Reagent |
Volume |
x11 |
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Expected sizes = 100 bp
1. YFP2
2. RFP1
3. GAPDH
4. MV2
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10 μL/lane, 1% agarose
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| cDNA |
0.5 |
---
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| 10μM primer mix |
2.0 |
22.0
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| 2x GoTaq Green |
10.0 |
110.0
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| dH2O |
7.5 |
82.5
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20.0 |
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--> PCR: 95°C/ 3 min.; [95°C/ 30 sec, 57°C/ 30 sec, 72°C/ 30 sec] x 30; 72°C/ 3 min.; 4°C/ ∞
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Project name
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