What s RNA extraction protocol with the trisol LS for the cell?

Salam Asma, here goes a brief protocol-

First lyse your samples by pipetting with Add 0.75 mL of TRIzolTM LS Reagent per 0.25 mL of sample.

Centrifuge the lysate for 5 minutes at 12,000 × g at 4°C, transfer the top clear supernatant to a new tube.

Incubate for 5 minutes at Room Temp. to permit complete dissociation of the nucleoproteins complex.

Add 0.2 mL of chloroform per 0.75 mL of TRIzolTM LS Reagent used for lysis. Incubate for 2–3 minutes.

Centrifuge the sample for 15 minutes at 12,000 × g at 4°C. The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase.

Transfer the aqueous phase containing the RNA to a new tube. Don't ever touch the interphase step, if you touch, then repeat step 5.

For purification-

7. Add 0.5 mL of isopropanol to the aqueous phase, per 0.75 mL of TRIzolTM LS Reagent used for lysis.Incubate for 10 minutes. Centrifuge for 10 minutes at 12,000 × g at 4°C.

8. Discard the supernatant and resuspend the pellet in 1 mL of 75% ethanol. Centrifuge for 5 minutes at 7500 × g at 4°C.

9. Discard the supernatant (Total RNA precipitate forms a white gel-like pellet at the bottom of the tube).

10. Vacuum or air dry the RNA pellet for 5–10 minutes.

11. Resuspend the pellet in 20–50 μL of RNase-free water, by pipetting up and down.

12. Incubate in a heat block set at 60°C for 10 minutes. Proceed to downstream applications, or store the RNA at –70°C.

Good Luck :)

Asma Vafadar

thank you for help me but in protocol trizol LS mention:

TRIzol™ LS Reagent is designed for processing liquid samples (blood and virus preparations, for example). Do not use TRIzol™ LS Reagent undiluted with solid samples. Processing solid samples with TRIzol™ LS Reagent results in decreased yield ..

but cell that isnt liquid .how can i use it for cell?

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