-
Washing of Mag-Net™
Oligo dT Matrix:
-
Add
100 µL
resuspended Mag-Net Oligo dT Matrix to a sterile RNase-free
microcentrifuge tube. Place the tube in the Mag-Net™ Tube Stand for 30
seconds and remove the clear supernatant. Remove the tube from the Mag-Net
Tube Stand, add 100 µL
of the Binding Reagent, and gently vortex to resuspend. Leave the Mag-Net
Oligo dT Matrix resuspended until the Total RNA sample is prepared.
-
Preparation of the
Total RNA Sample:
-
Use 75
µg
of Total RNA in a final sample volume of 100 µL
to achieve optimal annealing between the mRNA and the Mag-Net Oligo dT
Matrix. If a sample is more concentrated than 75 µg/100 µL,
use nuclease-free water for dilution up to 100 µL.
For a sample that is more dilute than 75µg/100 µL,
simply follow the protocol without modifying the sample. Add 100 µL
of Binding Reagent to the Total RNA sample and incubate at 65°C for 2
minutes.
-
mRNA Isolation
Protocol:
-
Remove the
Binding Reagent from the pre-washed Mag-Net Oligo dT Matrix (Step 1) by
inserting the tube in the Mag-Net Tube Stand for 30 seconds. Remove the
supernatant and add 100 µL
of Binding Reagent.
-
Add
the 200 µL
of Total RNA sample (Step 2) to the Oligo dT Matrix resuspension. Mix the
tube by inversion or rotation for 3-5 minutes at room temperature to allow
annealing. Place the tube in the Mag-Net Tube Stand for 30 seconds and
carefully remove the supernatant.
-
Wash the magnetic matrix with 200 µL
of the Wash Reagent, mix thoroughly, and carefully remove all traces of
the supernatant using the Mag-Net Tube Stand. Repeat a second time.
-
Add 20 µL
of Elution Reagent to the tube, gently vortex, and pulse centrifuge to
pool the matrix suspension to the bottom of the tube. Incubate at 65°C
for 2 minutes. Place the tube in the Mag-Net Tube Stand for 30 seconds,
then transfer the supernatant containing the mRNA to a sterile, RNase-free
microcentrifuge tube. The final elution step may also be performed using
sterile, deionized water or loading buffer containing formamide.
