Galenvs Magneti Total Rna Extraction Kit User Guide

magneti
Total RNA Extraction Kit
Quick Start Guide
Part A RNA Extraction

magneti Total RNA Extraction Kit

1. Thaw Paxgene tube for 2 hours at room temperature.
   Spin down PAXgene® tube at 5000g for 10 mins. Discard supernatant.
2. Resuspend pellet in 1.85ml nuclease-free, sterile water.
   Transfer suspension to microfuge tube.
3. Spin down tube again at 5000g for 10 mins.
   Discard supernatant.
4. Resuspend pellet in 300l of Suspension Buffer.
5. Add 40 ul of Proteinase K.
6. Add 300ul of Binding Buffer A and mix by pipetting. Vortex tube for 15 minutes with a vortex shaker at moderate speed.
7. Add 40ul of Binding Beads A and mix by pipetting.
   Vortex tube for 10 minutes with a vortex shaker.
8. Place tube on magnetic rack for 2 mins to capture the RNA-bead complex.
   Discard supernatant.
9. Resuspend beads in 600ul Wash Buffer A1.
   Mix by vortex shaker for 5 mins at moderate speed.
   Place tube on magnetic rack for 2 mins to capture beads, then discard supernatant.
10. Resuspend beads in 600ul Wash Buffer A2.
    Mix by pipetting 20x.
    Place tube on magnetic rack for 2 mins, then discard supernatant.
11. Repeat washing step with 600ul Wash Buffer A2.
    Place tube on magnetic rack for 2 mins, then discard supernatant.
12. Dry beads on magnetic rack for 2 mins after the third wash.
    Remove any wash buffer left at the bottom of tube at the end of drying step.
13. Add 90ul of Elution Buffer A.
    Pipette 20x and incubate for 5 mins.
    Place tube on magnetic rack for 5 mins.
14. Transfer supernatant to clean microfuge tube.
    For part B only 50ul of the elution is required.Proceed to part B DNase Treatment
15. Add 100ul of DNase Reaction Buffer to
    DNase pellet and then add 100ul of glycerol. Mix by gently inverting the tube.
    Reconstituted pellet must be stored at -20 °C.
16. For each RNA sample, prepare DNase buffer by adding 10ul of DNase prepared in the previous step to 40ul of DNase
    Reaction Buffer in a microfuge tube.
    Mix DNase with buffer by gently inverting the tube a few times.
17. Add 50ul of DNase buffer to 50ul of RNA sample in a microfuge tube.
18. Gently shake microfuge tube and incubate at room temperature for 25 minutes.
19. Mix 40ul of Binding Beads B with 400ul Binding Buffer B.
20. Add 400ul of Binding Bead and Binding Buffer mixture to microfuge tube and mix by pipetting.
21. Vortex for 5 mins in a vortex shaker.
22. Place tube on magnetic rack for 2 mins.
    Discard supernatant.
23. Resuspend beads in 600ul Wash Buffer B1.
    Mix by pipetting 20x.
24. Place tube on magnetic rack, wait for 2 minutes and discard supernatant.
25. Repeat washing step with Wash Buffer B1.
26. Dry beads on magnetic rack for 2 mins after the second wash.
    Remove any wash buffer left at the bottom of tube at the end of drying step.
27. Add 50ul of Elution Buffer B to tube.
    Mix by pipetting 20x and incubate for 5 mins.
28. Place tube on magnetic rack, wait for 5 mins and then transfer supernatant to clean tube.

Total RNA Extraction Kit
TRB0100-120TRB0250-12