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

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(01/27/14)
 
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==01/27/14==
==01/27/14==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
-
* qRT-PCR experiment - PlateK562_1/ U2OS
+
* U2OS RT-PCR - RNA extraction & cDNA synthesis
 +
 
----
----
-
'''qRT-PCR - Plate K562_1/ U2OS'''
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'''RNA Extraction'''
-
* Protocol: http://openwetware.org/wiki/Haynes:UPLassay
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* Use combined TRIzol/ spin column metod. Use RNeasy Mini Kit.
-
* Use layout K5621 on U2OS samples
+
-
** File: Projects > Cancer PcTF > RT-PCR data > '''Plate_layouts_Carly.xlsx'''
+
 +
Samples (TRIzol-lysed U2OS samples from -80°C freezer)
 +
# Tube 1-A (+PcTF)
 +
# Tube 1-A (+PcTF)
 +
# Tube 4-A (mock)
 +
# Tube 4-A (mock)
-
{|
+
Procedure notes (based on Carly's notes from [http://openwetware.org/wiki/Haynes_Lab:Notebook/Synthetic_Biology_and_Bioinformatics_for_Predictable_Control_of_Therapeutic_Gene2/2013/01/07 1/10/13])<br>
-
| align="center" style="background:#f0f0f0;"| &nbsp;
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'''TRIzol steps'''
-
| align="center" style="background:#f0f0f0;"|'''Template'''
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* Clean all work surfaces and pipettors with RNase-zap.
-
| align="center" style="background:#f0f0f0;"|'''Gene Target'''
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* Thaw TRIzol-lysed cells at room temp
-
|-
+
* Under fume hood: Add '''100 μL chloroform''' to each sample
-
| Rxn 1:||treated cells||KIT, primers 13/14
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* Shake vigorously (by inverting tubes) for 20 sec. Let sit at room temp. for 2 min.
-
|-
+
* Centrifuge in cold room (4°C) for 15 min @ 12,000G
-
| Rxn 2:||treated cells||TNFRSF11A, primers 15/16
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* Pipet off clear phase from organic phase (pink) into new tube (AVOID THE PINK PHASE)
-
|-
+
* Check volume of clear phase (in new tubes). Add same volume of 70% ethanol, RNase-free. Mix and incubate at room temp (refer to RNeasy protocol)
-
| Rxn 3:||treated cells||EGFR, primers 17/18
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'''Spin-column steps'''
-
|-
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* Open up one pink, sealed spin-column (with attached cap) per sample, label it, and place into collection tube(s).
-
| Rxn 4:||treated cells||WT1, primers 19/20
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* Transfer RNA/ethanol mix to each spin column.
-
|-
+
* Spin tubes at top speed for 30 sec (room temp).
-
| Rxn 5:||treated cells||HLF, primer 21/22
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* Discard flow-through liquid and add 700μL of RW1 Buffer to the spin column.
-
|-
+
* Spin tubes at top speed for 30 sec (room temp).
-
| Rxn 6:||treated cells||BCL6, primer 23/24
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* Put collection vial in a fresh collection tube and discard the old one.
-
|-
+
* --> Add 500μL of RBE Buffer to the spin column.
-
| Rxn 7:||treated cells||mCh
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* Spin tubes at top speed for 30 sec (room temp) then discard the liquid flow-through.
-
|-
+
* Repeat the above two steps again. -->
-
| Rxn 8:||treated cells||ref. gene, GAPD
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* Discard the flow-through and spin again.
-
|-
+
* Transfer liquid to a fresh 1.5 mL tube (RNase-free) and add 30 μL of RNase-free H<sub>2</sub>O.
-
| Rxn 9:||untreated cells||KIT, primers 13/14
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* Let tubes sit for 1 min at room temp.
-
|-
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* Spin tubes for 1 min (room temp).
-
| 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
+
-
|-
+
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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
+
-
|-
+
-
| Rxn 24:||no template||ref. gene, GAPD
+
-
|}
+
-
'''Primer/Probe Master Mixes (8 total)'''
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'''Measure RNA Concentration'''
 +
* Use the BioTek Take3 plate/ reader
 +
 
{|
{|
-
| align="center" style="background:#f0f0f0;"|'''Reagent'''
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| Sample || OD 260 || 260/280 || ng/μL
-
| 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)'''
+
|-
|-
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| 2x LC480 Probes Master||(7.5 μL)||75||75
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| 1. 1-A (+PcTF) || 1.007 || 2.1 || 805.8
|-
|-
-
| 20 μM Forward primer||(0.3 μL)||3||3.0 GAPD primers*
+
| 2. 1-A (+PcTF) || 0.843 || 2.1 || 674.0
|-
|-
-
| 20 μM Reverse primer||(0.3 μL)||3||---
+
| 3. 4-A (+PcTF) || 0.992 || 2.1 || 793.9
|-
|-
-
| 10 μM UPL probe||(0.3 μL)||3||3.0 GAPD UPL probe*
+
| 4. 4-A (+PcTF) || 0.959 || 2.1 || 767.0
-
|-
+
-
| PCR H2O||(0.1 μL)||1||4
+
-
|-
+
-
| Total vol.||(8.5 μL)||85||85
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|}
|}
 +
* Keep on ice. Store stock RNA at -80°C after samples are used for cDNA synthesis.
-
'''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||---
 
-
|-
 
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| 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'''
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'''cDNA Synthesis'''
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* First of each triplicate well, add 25.5 Primer/Probe MM + 19.5 Template MM
+
* Use Superscript III kit (freezer)
-
* Transfer 15 μL from well 1 to wells 2 and 3 for each set
+
* Pre-heat the heat block to 65°C. Use the PCR strip-tube-compatible block
 +
 
 +
Procedure notes
 +
* Kit components kept on ice: RNase H, SS III RT, and RNase out
 +
* Kit components thawed at Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT
 +
 
 +
* Label four 0.2 mL PCR strip-tubes
 +
* Make 8 μL solutions that contain 2.0 μg RNA
 +
** vol stock RNA (μL) = 2000 ng/ [RNA] from table
 +
** vol H<sub>2</sub>O = 8.0 μL - vol stock RNA
 +
* Make duplicates for each sample. Use RNA stocks #1 and #3.
 +
# Reaction 1: 2.5 μL Stock RNA #1 + 5.5 μL H<sub>2</sub>O = 2.5ug
 +
# Reaction 2: 2.5 μL Stock RNA #1 + 5.5 μL H<sub>2</sub>O = 2.5ug
 +
# Reaction 3: 2.5 μL Stock RNA #3 + 5.5 μL H<sub>2</sub>O = 2.5ug
 +
# Reaction 4: 2.5 μL Stock RNA #3 + 5.5 μL H<sub>2</sub>O = 2.5ug
 +
 
 +
'''Part 1'''
 +
* Add 1μL of primer (Oligo dT)
 +
* Add 1μL of dNTP
 +
* Total volume is 10 μL
 +
* Incubate for 5 min @ 65°C
 +
* Immediately place on ice and incubate for 1 min.
 +
 
 +
'''Part 2'''
 +
* Make a master mix for 4 rxns:
 +
** Single reaction = 2 μL RT Buffer, 4 μL MgCl2, 2 μL DTT, 1 μL RNaseOut, 1 μL SuperScript III
 +
** 4 reactions = 8 μL RT Buffer, 16 μL MgCl<sub>2</sub>, 8 μL DTT, 4 μL RNaseOut, and 4 μL SuperScript III
 +
* Carefully add 10μL of MM to each rxn tube
 +
* Mix tubes gently by flicking, centrifuge for a few seconds
 +
 
 +
'''Part 3'''
 +
* PCR Machine, set up as follows:
 +
# Stage 1: 50 min @ 50°C
 +
# Stage 2: 5 min @ 85°C
 +
# Stage 3: Infinity @ 4°C
 +
* 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
 +
* Store at -20°C
 +
 

Current revision

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

  • U2OS RT-PCR - RNA extraction & cDNA synthesis



RNA Extraction

  • Use combined TRIzol/ spin column metod. Use RNeasy Mini Kit.

Samples (TRIzol-lysed U2OS samples from -80°C freezer)

  1. Tube 1-A (+PcTF)
  2. Tube 1-A (+PcTF)
  3. Tube 4-A (mock)
  4. Tube 4-A (mock)

Procedure notes (based on Carly's notes from 1/10/13)
TRIzol steps

  • Clean all work surfaces and pipettors with RNase-zap.
  • Thaw TRIzol-lysed cells at room temp
  • Under fume hood: Add 100 μL chloroform to each sample
  • Shake vigorously (by inverting tubes) for 20 sec. Let sit at room temp. for 2 min.
  • Centrifuge in cold room (4°C) for 15 min @ 12,000G
  • Pipet off clear phase from organic phase (pink) into new tube (AVOID THE PINK PHASE)
  • Check volume of clear phase (in new tubes). Add same volume of 70% ethanol, RNase-free. Mix and incubate at room temp (refer to RNeasy protocol)

Spin-column steps

  • Open up one pink, sealed spin-column (with attached cap) per sample, label it, and place into collection tube(s).
  • Transfer RNA/ethanol mix to each spin column.
  • Spin tubes at top speed for 30 sec (room temp).
  • Discard flow-through liquid and add 700μL of RW1 Buffer to the spin column.
  • Spin tubes at top speed for 30 sec (room temp).
  • Put collection vial in a fresh collection tube and discard the old one.
  • --> Add 500μL of RBE Buffer to the spin column.
  • Spin tubes at top speed for 30 sec (room temp) then discard the liquid flow-through.
  • Repeat the above two steps again. -->
  • Discard the flow-through and spin again.
  • Transfer liquid to a fresh 1.5 mL tube (RNase-free) and add 30 μL of RNase-free H2O.
  • Let tubes sit for 1 min at room temp.
  • Spin tubes for 1 min (room temp).


Measure RNA Concentration

  • Use the BioTek Take3 plate/ reader
Sample OD 260 260/280 ng/μL
1. 1-A (+PcTF) 1.007 2.1 805.8
2. 1-A (+PcTF) 0.843 2.1 674.0
3. 4-A (+PcTF) 0.992 2.1 793.9
4. 4-A (+PcTF) 0.959 2.1 767.0
  • Keep on ice. Store stock RNA at -80°C after samples are used for cDNA synthesis.


cDNA Synthesis

  • Use Superscript III kit (freezer)
  • Pre-heat the heat block to 65°C. Use the PCR strip-tube-compatible block

Procedure notes

  • Kit components kept on ice: RNase H, SS III RT, and RNase out
  • Kit components thawed at Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT
  • Label four 0.2 mL PCR strip-tubes
  • Make 8 μL solutions that contain 2.0 μg RNA
    • vol stock RNA (μL) = 2000 ng/ [RNA] from table
    • vol H2O = 8.0 μL - vol stock RNA
  • Make duplicates for each sample. Use RNA stocks #1 and #3.
  1. Reaction 1: 2.5 μL Stock RNA #1 + 5.5 μL H2O = 2.5ug
  2. Reaction 2: 2.5 μL Stock RNA #1 + 5.5 μL H2O = 2.5ug
  3. Reaction 3: 2.5 μL Stock RNA #3 + 5.5 μL H2O = 2.5ug
  4. Reaction 4: 2.5 μL Stock RNA #3 + 5.5 μL H2O = 2.5ug

Part 1

  • Add 1μL of primer (Oligo dT)
  • Add 1μL of dNTP
  • Total volume is 10 μL
  • Incubate for 5 min @ 65°C
  • Immediately place on ice and incubate for 1 min.

Part 2

  • Make a master mix for 4 rxns:
    • Single reaction = 2 μL RT Buffer, 4 μL MgCl2, 2 μL DTT, 1 μL RNaseOut, 1 μL SuperScript III
    • 4 reactions = 8 μL RT Buffer, 16 μL MgCl2, 8 μL DTT, 4 μL RNaseOut, and 4 μL SuperScript III
  • Carefully add 10μL of MM to each rxn tube
  • Mix tubes gently by flicking, centrifuge for a few seconds

Part 3

  • PCR Machine, set up as follows:
  1. Stage 1: 50 min @ 50°C
  2. Stage 2: 5 min @ 85°C
  3. Stage 3: Infinity @ 4°C
  • 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
  • Store at -20°C



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