05/05/10
- ✓ FlpE: cultures (2 from 20 μg/mL Amp plate, 2 from 100 μg/mL Amp plate)
- ✓ RT-PCR: Pc-ATF 4-day dox set, p14ARF & KRT17
- ✓ Senescence assay cultures: Expand 132-4, -6 to 10 cm plates; KAH126-1 not growing well (freeze down current culture, PS7, on next passage); thaw fresh U2OS Flp-in T-REx
RT-PCR
> Continued from 5/04/10: semi-qRT-PCR
--> cDNA templates
- KAH126-1
- KAH126-1(+dox)
- KAH127-4
- KAH127-4(+dox)
- KAH128-8
- KAH128-8(+dox)
- KAH129-4
- KAH129-4(+dox)
- KAH130-2
- KAH130-2(+dox)
- KAH131-9
- KAH131-9(+dox)
- KAH132-8
- KAH132-8(+dox)
- KAH133-1
- KAH133-1(+dox)
- FTRx
- FTRx(+dox)
--> Primers; cDNA dilution
- p14ARF(6); 1:1
- KRT17(14A); 1:100
Reagent |
Volume |
Primer mix (x20)
|
15 μL/lane; 2% agarose
|
cDNA |
0.5 |
---
|
10μM primers |
1.0 |
20.0
|
2x GoTaq Green |
10.0 |
200.0
|
dH2O |
8.5 |
170.0
|
|
20.0 μL
|
|
--> Quantitate KRT17 using Image J
|