User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/12/23

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Contents

Rotifer mRNA extraction

Materials

  • 300 ul lysis buffer
  • 50 ul Lysis/Binding buffer
  • 1200 ul washing buffer A
  • 600 ul washing buffer B
  • 10 ul elution buffer

Lyse sample

  • pellet several hundred A. ricciae rotifers (400g 8 min), wash with PBS, and pellet
  • add 300 ul lysis buffer, pipette up and down

Prepare beads

  • add 10 ul of beads into 1.5ml RNAse-free tube
  • pellet beads 30s with magnet
  • wash with 50ul Lysis/Binding buffer

mRNA isolation

  • remove Lysis/Binding buffer from beads
  • add sample lysate
  • incubate with mixing for 3-5 min
  • place vial on magnet for 2 min, remove supernatant
  • wash beads 2x with 600 ul washing buffer A
  • wash 2x with 300 ul washing buffer B
  • elute by adding 10 ul of Tris-Hcl solution and incubating at 65C for 2 minutes
  • immediately pellet the beads, and transfer supernatant on ice
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