mRNA Purification Protocol (Qiagen)
Joanna Liliental
TASC
1/30/2012
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Heat Oligotex Suspension to 37C in a water-bath.
Mix by vortexing.
Place at RT.
Heat OEB to 70C.
Determine the amount of RNA by spec.
Heat OBB to 37C to dissolve ppt. Then place at RT.
All to be done at RT.
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Pipet RNA into an RNase-free 1.5 microcentrifuge tube.
Adjust volume with Rnase-free H2O as shown in Table 4 (up to 250ul).
Add OBB (250ul) and Oligotex Suspension (15ul) as shown in Table 4.
Mix the contents by pipeting or flicking tube.
Incubate sample for 3 min at 70C to disrupt 2o structure of RNA.
Remove sample form heat block.
Place sample at RT for 10 min (hybridize dT30 to polyA of mRNA).
Pellet the Oligotex: mRNA complex by spinning 2 min @ 10,000rpm.
Remove supe by pipetting (leave approx. 50ul supe behind to prevent loss of resin) and
SAVE.
Resuspend Oligotex:mRNA pellet in 400ul OW2 by vortexing or pipeting.
Pipet onto small spin column placed in a 1.5ml tube.
Centrifuge for 1 min @ 10,000 rpm.
Transfer spin column to a new Rnase free 1.5ml centrifuge tube.
Apply 400ul OW2 to column.
Centrifuge 1 min @ 10,000rpm.
Discard flow through.
Transfer spin column to a new tube.
Place tube in 70C (with spin column, Oligotex and sample).
Pipet 20-100ul 70C OEB onto the column.
Pipet up and down 3-4 times to resuspend the resin.
Centrifuge 1 min @ 10,000 rpm.
REPEAT 19-21 (could use 1st eluate for 2nd elution).