Set up
- TRIzol added to treated/untreated cell pair at time points
| 3U |
3 day untreated
|
| 6T |
6 day treated
|
| 6U |
6 day untreated
|
| 10T |
10 day treated
|
| 10U |
10 day untreated
|
RNA Extraction
- Use combined TRIzol/ spin column metod. Use RNeasy Mini Kit.
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 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.
- Reaction 1: 2.5 μL Stock RNA #1 + 5.5 μL H2O = 2.5ug
- Reaction 2: 2.5 μL Stock RNA #1 + 5.5 μL H2O = 2.5ug
- Reaction 3: 2.5 μL Stock RNA #3 + 5.5 μL H2O = 2.5ug
- 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:
- 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
|
Cell Fate Switch by Synthetic Transcription Factors
