05/03/10
- ✓ RT-PCR: 4-day dox Pc-ATF lines
- ✓ Bact. cultures: FlpE (4)
- ✓ Senescence assay cultures: expand/split confluent cultures
- ✓ Pc-ATF negative ctrl lines: Pick colonies (96-well plates, 32 colonies/line)
RT-PCR
> Continued from 4/29/10: semi-qRT-PCR
--> Use 1:100 dilution for mCh primer (10,000 might be too low)
--> 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
- p15INK(5); 1:1
- p16INK4a(7B); 1:1
- mCh f1/r1; 1:100
- GAPDH(21A); 1:10,000
Reagent |
Volume |
Master mix (x20)
|
15 μL/lane; 2% agarose
|
cDNA |
0.5 |
---
|
10μM primers |
1.0 |
10.0
|
2x GoTaq Green |
10.0 |
100.0
|
dH2O |
8.5 |
85.0
|
|
20.0 μL
|
|
--> Looks interesting; 126-1 appears to increase p16INK
--> Redo GAPDH controls (21A and 21B primers) with 1:1000 dilution
|