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05/21/14
- qRT-PCR: Run "K562_2" plate for SKNSH and U2OS
- Order: Roche UPL human probe #1
RT-PCR: QC_plate, all cDNA batches
- System: Roche LC480 (machine & reagents)
- Summary: Complementary experiment; Look at expression of genes identified as unique targets for K562 cells
- Experiment file name: DualColorProbe_UPL_Haynes052114
- Wells per Target Gene: 4 cDNAs x 3 reps = 12
- Wells per cDNA Template: 7 genes x 3 reps = 21
- cDNA dilution: 1:10 for all reactions
- Plate layout: Plate_layouts_Carly.xlsx/K562_2
Target Genes & Oligos:
- PBK, UPL-21, Plate1 A7 (100 μM mixed F/R primers)
- MAP4K1, UPL-1, Plate1 A8
- RIPK2, UPL-3, Plate 1 A9
- FUK, UPL-10, Plate1 A10
- LMTK3, UPL-1, Plate 1 A11
- PIP5KL1, UPL-19, Plate 1 A12
- PRKAA1, UPL-2, Plate1 B1
Primer/Probe Master mixes (7 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:
- SKNSH_E001 (E = experimental, +PcTF)
- SKNSH_C001 (C = control, no PcTF)
- U2OS_E002
- U2OS_C002
Template master mixes (4 tubes)
| Reagent
|
(Single well)
|
cDNA Template (x21.5)
|
no-template control (x0)
|
| diluted Batch# cDNA |
(2.0 μL) |
43.0 |
0.00
|
| PCR H2O |
(4.5 μL) |
96.8 |
0.00
|
| Total vol. |
(6.5 μL) |
139.8 |
0.00
|
|
Pc-TF Genomics
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