User:Karmella Haynes/Notebook/PcTF Genomics/2014/01/27: Difference between revisions

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==01/27/14==
==01/27/14==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* qRT-PCR experiment - PlateK562_1/ U2OS


----
----
'''Line item 1'''<br>
'''qRT-PCR - Plate K562_1/ U2OS'''
> Samples
* Protocol: http://openwetware.org/wiki/Haynes:UPLassay
* Use layout K5621 on U2OS samples
** File: Projects > Cancer PcTF > RT-PCR data > '''Plate_layouts_Carly.xlsx'''


{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
 
|-valign="top"
{|
| <u>Reagent</u> || <u>Volume</u>
| align="center" style="background:#f0f0f0;"| &nbsp;
| align="center" style="background:#f0f0f0;"|'''Template'''
| align="center" style="background:#f0f0f0;"|'''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
|-
|-
| reagent 1 || # μL
| Rxn 18:||no template||TNFRSF11A, primers 15/16
|-
|-
| reagent 2 || #
| Rxn 19:||no template||EGFR, primers 17/18
|-
|-
| reagent 3 || #
| 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)'''
{|
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''(Single well)'''
| align="center" style="background:#f0f0f0;"|'''Gene Target 1 - 7 (x10)'''
| align="center" style="background:#f0f0f0;"|'''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 1:100 dilution of cDNA for GAPD
{|
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''(Single well)'''
| align="center" style="background:#f0f0f0;"|'''treated cDNA Template (x25)'''
| align="center" style="background:#f0f0f0;"|'''untreated cDNA Template (x25)'''
| align="center" style="background:#f0f0f0;"|'''no Template (x25)'''
|-
|-
| reagent 4 || #
| diluted cDNA||(2.0 μL)||50||50||---
|-
|-
| dH<sub>2</sub>O || #
| PCR H2O||(4.5 μL)||112.5||112.5||162.5
|-
|-
| &nbsp; || # μL
| Total vol.||(6.5 μL)||162.5||162.5||162.5
|}
|}


--> Reaction conditions


'''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




Revision as of 17:04, 24 January 2014

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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 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 1:100 dilution of cDNA for GAPD
Reagent (Single well) treated cDNA Template (x25) untreated cDNA Template (x25) no Template (x25)
diluted cDNA (2.0 μL) 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



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