DynabeadsmRNA DIRECT Micro Kit Isolate mRNA for reverse transcription and PCRCat. no. 610.21Initial Check-List?All buffers, except the 10 mM Tris-HCl, should be brought to room temperature prior to use. The 10 mM Tris-HCl should be stored on ice or 2-8 C prior to use.?Ensure that the DynabeadsOligo (dT)25have been fully resuspended before use. Resuspend by brief vortexing or careful pipetting.?Check that your Lysis/Binding Buffer has not precipitated. If any precipitation is observed, warm to room temperature and shake to full resuspension.?A rapid lysis in Lysis/Binding Buffer is critical for obtaining un-degraded mRNA. Thawing of frozen material prior to lysis must be avoided.?When working with cells isolated with cell-specific Dynabeads, make sure all the cell-specific Dynabeads are removed from the lysate before adding Dynabeads Oligo (dT)25?Prepare your reverse transcription reaction mix prior to mRNA isolation and keep on ice.?We recommend to immediately use the Dynabeads- mRNA complex for reverse transcription and PCR.?Use sterile, RNase-free microtubes and pipette tips.?Wear disposable gloves and change them frequently.?Refer to the package insert for detailed protocols.Mix 5 minutes on a rollerfor mRNA to hybridize to the oligo-dT on theat room-temperature beads.Place on magnet and remove supernatant. Add 100 l Washing Buffer A and resuspend by careful pipetting.Add 100 l Lysis/Binding Buffer to your starting sample and homogenize (e.g. by pipetting) until complete lysis is obtained.Transfer the lysate to a tube containing 20 l pre-washed Dynabeads Oligo (dT)25Wash once in 100 l Washing Buffer A and twice in.100 l Washing Buffer B. The magnet allows for easy washing and change of buffersResuspend the Dynabeads -mRNA complex in 100 l ice-cold Tris-HCl and store on ice for reverse transcription and downstream applications. (Elution is optional at this step.)Immediately prior to adding the reverse transcription PCR-mix to the , apply the magnet and remove the supernatant.Dynabeads -mRNA complexThe Dynabeads -mRNA complex is now ready for reverse transcription and PCR.TTTTTTTTTTTTRev. no. 001