User:David K. Barclay/Notebook/Controlling Pancreas Cell Fate Using Transcription Factors/2014/04/14

Cell Fate Switch by Synthetic Transcription Factors

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RNA extraction RNeasy spin column after TRIzol prep See following protocol: http://openwetware.org/wiki/Haynes:TRIzol_RNeasy 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 Protocol taken from Dr. Haynes 01/27/2014 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