A simple & Detailed Procedure for RNA Isolation from Mammalian Cell Lines using Trizol Reagent

  • Harvested cells can be stored in -80 degree Celsius for less than a week before RNA isolation or right after harvest.
  • Add 400 µl Trizol to lyse the cell and incubate at Room Temperature for 5, minutes.
  • Add 80 µl chloroform (0.2ml per 1ml Trizol)
  • Vortex the mixture for 15 secs and incubate at Room Temperature for 10 minutes
  • Centrifuge at 12000rpm / 4 degree Celsius / 15 minutes
  • Transfer the aqueous phase to the new clean tube and add equal volume of isopropanol
  • Mix well (short vortex)
  • Incubate at room temperature for 10 minutes.
  • Centrifuge at 12000 rpm / 10 minutes/ 4 degree Celsius
  • Remove isopropanol using pipette keeping the pellet safe
  • Add 400 µl of 75% ethanol and centrifuge at 7500 rpm/ 5 minutes/ 4 degree Celsius
  • Pipette out the ethanol keeping the pellet safe
  • Air dry the pellets for 5-10 minutes
  • Resuspend the pellet with 20 µl Nuclease Free water.
  • Measure the concentration of RNA isolated. (SPECTROPHOTOMETRIC ANALYSIS)

Binod G C

I'm Binod G C (MSc), a PhD candidate in cell and molecular biology who works as a biology educator and enjoys scientific blogging. My proclivity for blogging is intended to make notes and study materials more accessible to students.

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