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. Add 500 μL TRIzol. 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

  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)
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