User:Karmella Haynes/Notebook/PcTF Genomics/2014/05/29

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  • 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:

  1. HNF1B, UPL-18, Plate 1 D5 (100 μM mixed F/R primers)
  2. IKZF1, UPL-3, Plate 1 D6
  3. TAL1, UPL-1, Plate 1 D7
  4. FEV, UPL-2, Plate 1 D8
  5. RXRG, UPL-1, Plate 1 D9
  6. 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)93.750.00
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. 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 (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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