Haynes:TRIzol RNeasy

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RNA Miniprep - TRIzol/ RNeasy Sping Column Combo
by Karmella Haynes, 2013

Principle:

LYSE CELLS IN TRIZOL

1. Suspension cells: Add up to 1x10^6 cells to a 2.0 mL microfuge tube. Spin for 5 min. at 800 xg (~3000 rpm in a standard table top centrifuge) at room temp. Discard supernatant and add 500 μL TRIzol (per 1x10^6 cells). Mix by pipetting up and down. Incubate at room temperature for 5 min. Store at -80°C or go on to the next step.
2. Adherent cells: For cells grown to 100% confluency in a 6-well dish, aspirate off the growth medium and add 500 μL TRIzol to the cells. Incubate at room temperature for 5 minutes. Using a 1000 μL pipettor w/ disposable tip, scrape the bottom of the well to detach remaining cell debris, and transfer the total solution to a

EXTRACT RNA - PHASE SEPARATION

1. Add 200 μL chloroform per 1 mL TRIzol (100 μL per 500 μL TRIzol).
2. Make sure the lids are closed tightly. Mix by inverting for 20 seconds. Incubate at room temperature for 3 minutes.
3. Centrifuge samples at 12,000 xg for 15 minutes at 4°C. The mixture will separate into a clear upper aqueous phase, a cloudy interphase, and a pink lower organic (phenol/ chloroform) phase.
4. Carefully remove the tubes from the centrifuge. Do not disturb the phases.
5. Using a micropipette, carefully transfer the clear aqueous phase to a fresh RNase-free 1.5 mL tube. The aqueous phase volume should equal ~60% of the original TRIzol volume.
6. Slowly add an equal volume of RNase-free 70% EtOH to each sample.

SPIN-COLUMN RNA PURIFICATION

• Make sure that ethanol has been added to Buffer RPE.

1. Clean work area and micropipettors with RNaseZap.
2. Load up to 700 μL of sample* into an RNeasy spin column (seated in a collection tube). Spin for 30 seconds at top speed (≥ 8000 xg). Discard the flow-through. *Note: If the sample size is greater than 700, repeat this step, reusing the same column.
3. Add 700 μL Buffer RW1 into the column and spin for 30 seconds at top speed (≥ 8,000x g).
4. Transfer column into a new collection tube, add 500 μl Buffer RPE and spin 30 sec at ≥ 8,000x g. Discard flow-through.
5. Add another 500 μL Buffer RPE and spin for 2 minutes at top speed (≥ 8,000 xg). Discard flow-through.
6. Spin again (empty) for 1 minute at top speed (≥ 8,000 xg).
7. Transfer column into a fresh RNase-free 1.5 mL collection tube. Pipette 30-50 μL of RNase-free water directly onto the column membrane. Allow the sample to sit at RT for 1-2 min, and then spin 1 min at top speed (≥ 8,000 xg) to elute the RNA. Store at -80°C for up to one month.