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

Alien genes

Main project page Previous entry      Next entry

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