User:Karmella Haynes/Notebook/PcTF Genomics/2014/01/27: Difference between revisions
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* | * U2OS RT-PCR - RNA extraction & cDNA synthesis | ||
---- | ---- | ||
''' | '''RNA Extraction''' | ||
* | * Use combined TRIzol/ spin column metod. Use RNeasy Mini Kit. | ||
* Use | |||
** | 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> | |||
'''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 H<sub>2</sub>O. | |||
* 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 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 | |||
Revision as of 19:22, 14 May 2014
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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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