User:Karmella Haynes/Notebook/PcTF Genomics/2014/05/20

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05/20/14

  • qRT-PCR: Run "K562_1" plate for SKNSH 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 K562 cells
  • Experiment file name: DualColorProbe_UPL_Haynes052014
  • 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/K562_1


Target Genes & Oligos:

  1. MAPK14 - UPL-65, 1, 2 (20 μM separate primers)
  2. MAPK9 - UPL-57, 3, 4
  3. MAPK10 - UPL-1, 5, 6
  4. RET - UPL-3, 7, 8
  5. FGFR4 - UPL-1, 9, 10
  6. CDKN1A - UPL-1, 11, 12

Primer/Probe Master mixes (6 tubes)

Reagent (Single well) Target Gene (x12.5) Roche GAPD (x0)
2x LC480 Probes Master(7.5 μL)93.750.00
20 μM Forward primer(0.3 μL)3.750.00
20 μM Reverse primer(0.3 μL)3.750.00
10 μM UPL probe(0.3 μL)3.750.00
PCR H2O(0.1 μL)1.250.00
Total vol.(8.5 μL)106.250.00


cDNA Batches:

  1. SKNSH_E002 (E = experimental, +PcTF)
  2. SKNSH_C002 (C = control, no PcTF)
  3. U2OS_E002
  4. U2OS_C002

Template master mixes (4 tubes)

Reagent (Single well) cDNA Template (x18.5) no-template control (x0)
diluted Batch# cDNA(2.0 μL)37.00.00
PCR H2O(4.5 μL)83.30.00
Total vol.(6.5 μL)120.30.00



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