User:Karmella Haynes/Notebook/PcTF Genomics/2014/01/27: Difference between revisions
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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"| | |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 ✓ --> | ||
* U2OS RT-PCR - RNA extraction & cDNA synthesis | * U2OS RT-PCR - RNA extraction & cDNA synthesis | ||
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* Under fume hood: Add '''100 μL chloroform''' to each sample | * 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. | * Shake vigorously (by inverting tubes) for 20 sec. Let sit at room temp. for 2 min. | ||
* Centrifuge in cold room for 15 min @ 12,000G | * 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) | * 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) | * 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) | ||
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* Let tubes sit for 1 min at room temp. | * Let tubes sit for 1 min at room temp. | ||
* Spin tubes for 1 min (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. | |||
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'''cDNA Synthesis''' | '''cDNA Synthesis''' | ||
* Use Superscript III kit (freezer) | * Use Superscript III kit (freezer) | ||
* Pre-heat the heat block to 65°C. Use the PCR strip-tube-compatible block | |||
Procedure notes | Procedure notes | ||
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* Kit components thawed at Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT | * 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 | |||
Latest revision as of 23:39, 26 September 2017
Pc-TF Genomics | Main project page Previous entry Next entry | ||||||||||||||||||||
01/27/14
RNA Extraction
Samples (TRIzol-lysed U2OS samples from -80°C freezer)
Procedure notes (based on Carly's notes from 1/10/13)
Spin-column steps
cDNA Synthesis
Procedure notes
Part 1
Part 2
Part 3
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