RNA Isolation (RNeasy Micro Kit)

Contents:

 

Homogenise The Sample

For pelleted cells, loosen the cell pellet by thoroughly flicking the tube.

Add appropriate volume of RLT buffer (<5 X 10^6 cells = 350uL)

  • Homogenise the sample for 30 seconds using a rotor-stator homogeniser.
  • Add 1 volume 350uL 70% ethanol to homogenised lysate.
  • Mix thoroughly by pipetting.

DO NOT CENTRIFUGE. Continue immediately with the next step.

  • Apply 700uL at a time of the sample to an RNeasy MinElute Spin column in a 2mL collection tube (supplied). Close the tube gently, and centrifuge for 15 seconds at 10,000rpm. Discard the flow through.
  • Add 350uL RW1 Buffer to the RNeasy MinElute Spin column. Centrifuge for 15 seconds at 10,000rpm. Discard the flow through.

 

DNase on Column Treatment

  • Add 10uL DNase 1 stock solution to 70uL RDD Buffer. Mix gently by inverting the tube.
  • Pipet 80uL of the DNase 1 incubation mix directly onto the RNeasy MinElute silica-gel membrane, and incubate at room temperature for 15 minutes.
  • Pipet 350uL RW1 Buffer into the RNeasy MinElute Spin column, and centrifuge for 15 seconds at 10,000rpm. Discard flow through and collection tube.
  • Transfer the column to a new 2mL collection tube (supplied). Pipet 500uL RPE Buffer onto the column. Centrifuge for 15 seconds at 10,000rpm to wash the column. Discard the flow through.

 

Drying Step

  • Add 500uL 80% ethanol to the column. Centrifuge for 2 minutes at 10,000rpm to dry the silica-gel membrane, Discard the flow through and collection tube.
  •  Transfer the column to a new 2mL collection tube (supplied). Open the cap of the column and centrifuge in a microcentrifuge at maximum speed (13,000rpm) for 5 minutes. Discard the flow through and collection tube.

Elute the RNA:

  • Transfer column to a 1.5mL collection tube (supplied). Pipet 14uL RNase-free water directly onto the membrane. Centrifuge for 1minute at maximum speed (13,000rpm) to elute.