|
05/29/14
- Cell culture: KAH126, KAH154; passage, make new frozen stocks, set up 6-well plates for dox time-course
- qRT-PCR: Run "SKNSH_2" plate for K562 and U2OS
Cell culture
- KAH126 (PcTF) and KAH154 (delta-TF), ~1x10^7 cells each
- Passage 1:10 (1/10 of cells)
- Set up one 6-well plate for each (1/10 of cells) - will use these for dox time course & RT-PCR
- Use remainder for frozen stocks, 3 vials each (6/10 of cells)
qRT-PCR: Run "SKNSH_2" plate for K562 and U2OS
- System: Roche LC480 (machine & reagents)
- Summary: Complementary experiment; Look at expression of genes identified as unique targets for SKNSH cells
- Experiment file name: DualColorProbe_UPL_Haynes052914
- Wells per Target Gene: 4 cDNAs x 3 reps = 12
- Wells per cDNA Template: 6 genes x 3 reps = 18
- cDNA dilution: 1:10 for all reactions
- Plate layout: Plate_layouts_Carly.xlsx/SKNSH_2
Target Genes & Oligos:
- HNF1B, UPL-18, Plate 1 D5 (100 μM mixed F/R primers)
- IKZF1, UPL-3, Plate 1 D6
- TAL1, UPL-1, Plate 1 D7
- FEV, UPL-2, Plate 1 D8
- RXRG, UPL-1, Plate 1 D9
- MSX1, UPL-3, Plate 1 D10
Primer/Probe Master mixes (6 tubes)
| Reagent
|
(Single well)
|
Target Gene (x12.5)
|
Roche GAPD (x0)
|
| 2x LC480 Probes Master |
(7.5 μL) |
93.75 |
0.00
|
| 100 μM F/R primer mix |
(0.06 μL) |
0.75 |
0.00
|
| 10 μM UPL probe |
(0.3 μL) |
3.75 |
0.00
|
| PCR H2O |
(0.64 μL) |
8.00 |
0.00
|
| Total vol. |
(8.5 μL) |
106.25 |
0.00
|
cDNA Batches:
- K562_E001 (E = experimental, +PcTF)
- K562_C001 (C = control, no PcTF)
- U2OS_E002
- U2OS_C002
Template master mixes (4 tubes)
| Reagent
|
(Single well)
|
cDNA Template (x18.5)
|
no-template control (x0)
|
| diluted Batch# cDNA |
(2.0 μL) |
37.0 |
0.00
|
| PCR H2O |
(4.5 μL) |
83.3 |
0.00
|
| Total vol. |
(6.5 μL) |
120.3 |
0.00
|
|
Pc-TF Genomics
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