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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Alien genes
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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