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

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==01/27/14==
==01/27/14==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
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* qRT-PCR experiment - PlateK562_1/ U2OS
----
----
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'''Line item 1'''<br>
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'''qRT-PCR - Plate K562_1/ U2OS'''
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> Samples
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* Protocol: http://openwetware.org/wiki/Haynes:UPLassay
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* Use layout K5621 on U2OS samples
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** File: Projects > Cancer PcTF > RT-PCR data > '''Plate_layouts_Carly.xlsx'''
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{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
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|-valign="top"
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{|
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| <u>Reagent</u> || <u>Volume</u>
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| align="center" style="background:#f0f0f0;"| &nbsp;
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| align="center" style="background:#f0f0f0;"|'''Template'''
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| align="center" style="background:#f0f0f0;"|'''Gene Target'''
|-
|-
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| reagent 1 || # μL
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| Rxn 1:||treated cells||KIT, primers 13/14
|-
|-
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| reagent 2 || #
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| Rxn 2:||treated cells||TNFRSF11A, primers 15/16
|-
|-
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| reagent 3 || #
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| Rxn 3:||treated cells||EGFR, primers 17/18
 +
|-
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| Rxn 4:||treated cells||WT1, primers 19/20
 +
|-
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| Rxn 5:||treated cells||HLF, primer 21/22
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|-
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| Rxn 6:||treated cells||BCL6, primer 23/24
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|-
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| Rxn 7:||treated cells||mCh
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|-
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| Rxn 8:||treated cells||ref. gene, GAPD
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|-
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| Rxn 9:||untreated cells||KIT, primers 13/14
 +
|-
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| 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
 +
|-
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| Rxn 14:||untreated cells||BCL6, primer 23/24
 +
|-
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| Rxn 15:||untreated cells||mCh
 +
|-
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| Rxn 16:||untreated cells||ref. gene, GAPD
 +
|-
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| 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
 +
|-
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| Rxn 24:||no template||ref. gene, GAPD
 +
|}
 +
 
 +
 
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'''Primer/Probe Master Mixes (8 total)'''
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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;"|'''Gene Target 1 - 7 (x10)'''
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| align="center" style="background:#f0f0f0;"|'''Gene Target GAPD (x10)'''
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|-
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| 2x LC480 Probes Master||(7.5 μL)||75||75
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|-
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| 20 μM Forward primer||(0.3 μL)||3||3.0 GAPD primers*
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|-
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| 20 μM Reverse primer||(0.3 μL)||3||---
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|-
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| 10 μM UPL probe||(0.3 μL)||3||3.0 GAPD UPL probe*
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|-
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| PCR H2O||(0.1 μL)||1||4
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|-
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| Total vol.||(8.5 μL)||85||85
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|}
 +
 
 +
 
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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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| reagent 4 || #
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| diluted cDNA||(2.0 μL)||50||50||---
|-
|-
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| dH<sub>2</sub>O || #
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| PCR H2O||(4.5 μL)||112.5||112.5||162.5
|-
|-
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| &nbsp; || # μL
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| Total vol.||(6.5 μL)||162.5||162.5||162.5
|}
|}
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--> Reaction conditions
 
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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
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* Transfer 15 μL from well 1 to wells 2 and 3 for each set

Revision as of 19:04, 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
  • Transfer 15 μL from well 1 to wells 2 and 3 for each set



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