Difference between revisions of "User:Karmella Haynes/Notebook/PcTF Genomics/2014/02/05"

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span>
 
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Latest revision as of 22:42, 26 September 2017

Owwnotebook icon.pngPc-TF Genomics Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png


02/05/14

  • PcTF qRT-PCR - Plate K562_1/ U2OS

qRT-PCR - Plate K562_1/ U2OS


  Template Gene Target
Rxn 1: treated cells KIT, primers 13/14
Rxn 2: treated cells TNFRSF11A, primers 15/16
Rxn 3: treated cells EGFR, primers 17/18
Rxn 4: treated cells WT1, primers 19/20
Rxn 5: treated cells HLF, primer 21/22
Rxn 6: treated cells BCL6, primer 23/24
Rxn 7: treated cells mCh
Rxn 8: treated cells ref. gene, GAPD
Rxn 9: untreated cells KIT, primers 13/14
Rxn 10: untreated cells TNFRSF11A, primers 15/16
Rxn 11: untreated cells EGFR, primers 17/18
Rxn 12: untreated cells WT1, primers 19/20
Rxn 13: untreated cells HLF, primer 21/22
Rxn 14: untreated cells BCL6, primer 23/24
Rxn 15: untreated cells mCh
Rxn 16: untreated cells ref. gene, GAPD
Rxn 17: no template KIT, primers 13/14
Rxn 18: no template TNFRSF11A, primers 15/16
Rxn 19: no template EGFR, primers 17/18
Rxn 20: no template WT1, primers 19/20
Rxn 21: no template HLF, primer 21/22
Rxn 22: no template BCL6, primer 23/24
Rxn 23: no template mCh
Rxn 24: no template ref. gene, GAPD


Primer/Probe Master Mixes (8 total)

Reagent (Single well) Gene Target 1 - 7 (x10) Gene Target GAPD (x10)
2x LC480 Probes Master (7.5 μL) 75 75
20 μM Forward primer (0.3 μL) 3 3.0 GAPD primers*
20 μM Reverse primer (0.3 μL) 3 ---
10 μM UPL probe (0.3 μL) 3 3.0 GAPD UPL probe*
PCR H2O (0.1 μL) 1 4
Total vol. (8.5 μL) 85 85


Template Master Mixes

  • Use 1:10 dilution of cDNA for targets
  • Use 1:1000 dilution of cDNA for GAPD
Reagent (Single well) treated cDNA Template (x25) untreated cDNA Template (x25) no Template (x25)
diluted cDNA (2.0 μL) 50 50 ---
PCR H2O (4.5 μL) 112.5 112.5 162.5
Total vol. (6.5 μL) 162.5 162.5 162.5


96-well plate

  • First of each triplicate well, add 25.5 Primer/Probe MM + 19.5 Template MM
  • Transfer 15 μL from well 1 to wells 2 and 3 for each set