User:Karmella Haynes/Notebook/PcTF Genomics/2014/05/27

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05/27/14

  • qRT-PCR: Run "SKNSH_1" plate for K562 and U2OS



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 SKNSH cells
  • Experiment file name: DualColorProbe_UPL_Haynes052714
  • 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/SKNSH_1


Target Genes & Oligos:

  1. SFRP4, UPL-16, Plate 1 C10 (100 μM mixed F/R primers)
  2. TGFBR3, UPL-3, Plate 1 C11
  3. CHRNA3, UPL-1, Plate 1 C12
  4. EPHB4, UPL-21, Plate 1 D1
  5. IL13RA1, UPL-6, Plate 1 D2
  6. AXIN2, UPL-1, Plate 1 D3
  7. MAP3K1 , UPL-2, Plate 1 D4

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:

  1. K562_E001 (E = experimental, +PcTF)
  2. K562_C001 (C = control, no PcTF)
  3. U2OS_E002
  4. 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