User:Karmella Haynes/Notebook/PcTF Genomics/2014/01/27

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| Total vol.||(8.5 μL)||85||85
| Total vol.||(8.5 μL)||85||85
|}
|}
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'''Template Master Mixes'''
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* Use 1:10 dilution of cDNA for targets
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* Use 1:100 dilution of cDNA for GAPD
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{|
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| align="center" style="background:#f0f0f0;"|'''Reagent'''
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| align="center" style="background:#f0f0f0;"|'''(Single well)'''
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| align="center" style="background:#f0f0f0;"|'''treated cDNA Template (x25)'''
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| align="center" style="background:#f0f0f0;"|'''untreated cDNA Template (x25)'''
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| align="center" style="background:#f0f0f0;"|'''no Template (x25)'''
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|-
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| diluted cDNA||(2.0 μL)||50||50||---
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|-
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| PCR H2O||(4.5 μL)||112.5||112.5||162.5
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|-
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| Total vol.||(6.5 μL)||162.5||162.5||162.5
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|}
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* 96-well plate
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** First of each triplicate well, add 25.5 Primer/Probe MM + 19.5 Template MM

Revision as of 19:58, 24 January 2014

Pc-TF Genomics Main project page
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01/27/14

  • qRT-PCR experiment - PlateK562_1/ U2OS

qRT-PCR - Plate K562_1/ U2OS


  Template Gene Target
Rxn 1:treated cellsKIT, primers 13/14
Rxn 2:treated cellsTNFRSF11A, primers 15/16
Rxn 3:treated cellsEGFR, primers 17/18
Rxn 4:treated cellsWT1, primers 19/20
Rxn 5:treated cellsHLF, primer 21/22
Rxn 6:treated cellsBCL6, primer 23/24
Rxn 7:treated cellsmCh
Rxn 8:treated cellsref. gene, GAPD
Rxn 9:untreated cellsKIT, primers 13/14
Rxn 10:untreated cellsTNFRSF11A, primers 15/16
Rxn 11:untreated cellsEGFR, primers 17/18
Rxn 12:untreated cellsWT1, primers 19/20
Rxn 13:untreated cellsHLF, primer 21/22
Rxn 14:untreated cellsBCL6, primer 23/24
Rxn 15:untreated cellsmCh
Rxn 16:untreated cellsref. gene, GAPD
Rxn 17:no templateKIT, primers 13/14
Rxn 18:no templateTNFRSF11A, primers 15/16
Rxn 19:no templateEGFR, primers 17/18
Rxn 20:no templateWT1, primers 19/20
Rxn 21:no templateHLF, primer 21/22
Rxn 22:no templateBCL6, primer 23/24
Rxn 23:no templatemCh
Rxn 24:no templateref. 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)7575
20 μM Forward primer(0.3 μL)33.0 GAPD primers*
20 μM Reverse primer(0.3 μL)3---
10 μM UPL probe(0.3 μL)33.0 GAPD UPL probe*
PCR H2O(0.1 μL)14
Total vol.(8.5 μL)8585


Template Master Mixes

  • Use 1:10 dilution of cDNA for targets
  • Use 1:100 dilution of cDNA for GAPD
Reagent (Single well) treated cDNA Template (x25) untreated cDNA Template (x25) no Template (x25)
diluted cDNA(2.0 μL)5050---
PCR H2O(4.5 μL)112.5112.5162.5
Total vol.(6.5 μL)162.5162.5162.5


  • 96-well plate
    • First of each triplicate well, add 25.5 Primer/Probe MM + 19.5 Template MM



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