User:Karmella Haynes/Notebook/PcTF Genomics/2014/05/21

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  • 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:

  1. PBK, UPL-21, Plate1 A7 (100 μM mixed F/R primers)
  2. MAP4K1, UPL-1, Plate1 A8
  3. RIPK2, UPL-3, Plate 1 A9
  4. FUK, UPL-10, Plate1 A10
  5. LMTK3, UPL-1, Plate 1 A11
  6. PIP5KL1, UPL-19, Plate 1 A12
  7. 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)93.750.00
100 μM F/R primer mix(0.06 μL)0.750.00
10 μM UPL probe(0.3 μL)3.750.00
PCR H2O(0.64 μL)8.000.00
Total vol.(8.5 μL)106.250.00

cDNA Batches:

  1. SKNSH_E001 (E = experimental, +PcTF)
  2. SKNSH_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)43.00.00
PCR H2O(4.5 μL)96.80.00
Total vol.(6.5 μL)139.80.00

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