User:Karmella Haynes/Notebook/PcTF Genomics/2014/02/05: Difference between revisions
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* | * PcTF qRT-PCR - Plate K562_1/ U2OS | ||
---- | ---- | ||
''' | '''qRT-PCR - Plate K562_1/ U2OS''' | ||
> | * Protocol: http://openwetware.org/wiki/Haynes:UPLassay | ||
* Use layout K5621 on U2OS samples | |||
** File: Projects > Cancer PcTF > RT-PCR data > '''Plate_layouts_Carly.xlsx''' | |||
{| | |||
| | {| | ||
| align="center" style="background:#f0f0f0;"| | |||
| 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 | |||
|- | |||
| 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)''' | |||
{| | |||
| 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)''' | |||
|- | |||
| 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 | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Revision as of 14:29, 4 February 2014
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02/05/14
qRT-PCR - Plate K562_1/ U2OS
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