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| |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span> | | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span> |
| |style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} |
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| ==01/27/14== | | ==01/27/14== |
| <!-- Precede finished items with a checkmark ✓ --> | | <!-- Precede finished items with a checkmark ✓ --> |
| * qRT-PCR experiment - PlateK562_1/ U2OS
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| * U2OS RT-PCR - RNA extraction & cDNA synthesis | | * U2OS RT-PCR - RNA extraction & cDNA synthesis |
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| '''Part 3''' | | '''Part 3''' |
| * RT-PCR Machine, set up as follows: | | * PCR Machine, set up as follows: |
| # Stage 1: 50 min @ 50°C | | # Stage 1: 50 min @ 50°C |
| # Stage 2: 5 min @ 85°C | | # Stage 2: 5 min @ 85°C |
| # Stage 3: Infinity @ 4°C | | # Stage 3: Infinity @ 4°C |
| * Add 1μL: of RNase H | | * While PCR machine is running, change heat block temp to 37°C |
| | * After PCR program is finished, add 1 μL of RNase H to each reaction. Mix by flicking the tubes. |
| * Incubate @ 37°C for 20 min | | * Incubate @ 37°C for 20 min |
| * Store at -20°C | | * Store at -20°C |
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| ----
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| '''qRT-PCR - Plate K562_1/ U2OS'''
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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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| {|
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| | align="center" style="background:#f0f0f0;"|
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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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| |-
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| | Rxn 1:||treated cells||KIT, primers 13/14
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| |-
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| | Rxn 2:||treated cells||TNFRSF11A, primers 15/16
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| |-
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| | Rxn 3:||treated cells||EGFR, primers 17/18
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| |-
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| | Rxn 4:||treated cells||WT1, primers 19/20
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| |-
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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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| |-
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| | Rxn 10:||untreated cells||TNFRSF11A, primers 15/16
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| |-
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| | Rxn 11:||untreated cells||EGFR, primers 17/18
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| |-
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| | Rxn 12:||untreated cells||WT1, primers 19/20
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| |-
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| | Rxn 13:||untreated cells||HLF, primer 21/22
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| |-
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| | Rxn 14:||untreated cells||BCL6, primer 23/24
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| |-
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| | Rxn 15:||untreated cells||mCh
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| |-
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| | Rxn 16:||untreated cells||ref. gene, GAPD
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| |-
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| | Rxn 17:||no template||KIT, primers 13/14
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| |-
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| | Rxn 18:||no template||TNFRSF11A, primers 15/16
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| |-
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| | Rxn 19:||no template||EGFR, primers 17/18
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| |-
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| | Rxn 20:||no template||WT1, primers 19/20
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| |-
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| | Rxn 21:||no template||HLF, primer 21/22
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| |-
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| | Rxn 22:||no template||BCL6, primer 23/24
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| |-
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| | Rxn 23:||no template||mCh
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| |-
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| | Rxn 24:||no template||ref. gene, GAPD
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| |}
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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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| |-
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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
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| * Transfer 15 μL from well 1 to wells 2 and 3 for each set
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