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mRNA purification on oligo(dT) Dynabeads

mRNA purification Regeneration Solutions Notes  Comments

polyA RNA purification

• for each sample prepare 4x LoBind tubes:
  • 1.7ml — for hybridization reaction;
  • 1.7ml — for supernatant (Sup-tube);
  • 2x 0.5ml — for eluate;
• resuspend Dynabeads;


wash the necessary amount of Dynabeads:
NB! if several RNA samples are processed in parallel, prepare oligo(dT) Dynabeads for all of them at once.

number of samples:  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20
RNA Add Dynabeads W-buffer No quantity volume 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 total:



• take the total amount of Dynabeads:
• repeat two times:
    ◊  ◊   vortex beads 30 sec;
    ◊  ◊   magnetic rack 1 min & remove supernatant;
    ◊  ◊   add B-buffer:
  


1. prepare total RNA:
   • transfer RNA into LoBind tube;
   • place RNA at 65°C for 2 min, then - immediately on ice;
   • if necessary, add the necessary amount of 10mM Tris-HCl or B-buffer;



2. combine Dynabeads and RNA, mix carefully;
3. rotate 30-45 min at NT;
4. magnetic rack 1 min;
5. transfer supernatant into prepared Sup-tube;



6. 2x wash in W-buffer:
     ◊  ◊   resuspend beads in W-buffer;
     ◊  ◊   vortex beads, magnetic rack 1 min & transfer supernatant into Sup-tube;


preheat special(!) magnetic concentator;


7. two times elution:
     ◊  ◊   resuspend in 10µl of 10mM Tris-HCl;
     ◊  ◊   put for 2 min at 70°C;
     ◊  ◊   pulse spin, preheated magnetic rack 1 min & collect supernatant;
8. collect Dynabeads:
   • resuspend beads in original volume of S-buffer;
   • transfer into "Oligo(dT) REGENERATION" stock tube;
9. measure concentrations of both eluates;
10. snap freeze eluates and Sup-tube in liquid nitrogen, store at -70®C

Regeneration

1. resuspend used Dynabeads (original volume V);
   
2. 3x denaturation (and RNA degradation):
     ◊  ◊  ◊   resuspend in 1x V of 0.1M NaOH;
     ◊  ◊  ◊   vortex & pulse spin;
     ◊  ◊  ◊   incubate at 65°C, 2 min;
     ◊  ◊  ◊   magnetic rack 1 min & remove supernatant;
   
3. 4x wash (untill pH<8):
     ◊  ◊  ◊  ◊   resuspend in 1x V of S-buffer;
     ◊  ◊  ◊  ◊   vortex & pulse spin;
     ◊  ◊  ◊  ◊   magnetic rack 1 min & remove supernatant;
   
4. resuspend in 1x V of Storage Buffer, sign "regenerated oligo(dT) beads";
5. store at +4°C

Solutions

prepare solutions using Ambion's buffers



• B-buffer (Binding Buffer), store at +4°C

  5ml 10ml Tris-HCl, pH7.5 20mM 1M 100µl 200µl LiCl 1M 7.5M 670µl 1.33ml EDTA 2mM 0.5M 20µl 40µl H2O   mQ 4.22ml 8.43ml




• W-buffer (Washing Buffer) store at +4°C

  5ml 10ml Tris-HCl, pH7.5 10mM 1M 50µl 100µl LiCl 0.15M 7.5M 100µl 200µl EDTA 1mM 0.5M 10µl 20µl H2O   mQ 4.84ml 9.68ml




• Tris-HCl, 10mM pH7.5 store at +4°C

  5ml 10ml Tris-HCl, pH7.5 10mM 1M 50µl 100µl H2O   mQ 4.95ml 9.9ml




• 0.1M NaOH store at NT

  5ml 10ml NaOH 0.1M 1M 0.5ml 1ml H2O   mQ 4.5ml 9ml




• S-buffer (Storage Buffer), store at +4°C

  5ml 10ml Tris-HCl, pH7.5 250mM 1M 1.25ml 2.5ml EDTA 20mM 0.5M 200µl 400µl Tween20 10% 0.1% 50µl 100µl Sodium azide 10% 0.02% 10µl 20µl H2O   mQ 3.49ml 6.98ml

Notes

• Dynabeads Oligo(dT)₂₅:
  • concentration: 5 mg/ml
  • diameter: 2.8µm ± 0.2µm
  • surface area: 3-7m²/g
  • density: ~1.6g/cm³
  • magnetic mass susceptibility: 120 ± 25 x 10^-6 m³/kg
  • purification cost is 50µl of Dynabeads Oligo(dT)₂₅: 7.6€



• Total time is ~2h. Protocol should not be interrupted untill elution.



• After one round of purification ~50% of eluate is a ribosomal RNA (independent on washing quality). To decrease ribosomal RNA content it is necessary to repeat the purification.



• Critical importance is (i) not to dry out the beads, (ii) RNase free work. Time of incubation of Dynabeads with RNA may be prolongated for several hours without any negative effects (it is possible to set up a lot of hybridization reactions and then wash/elute them). It is unpractical to process more than 6 samples in parallel for washing and more than 2 samples for elution.



• Critical importance is (i) not to dry out the beads, (ii) RNase free work. Time of incubation of Dynabeads with RNA may be prolongated without any negative effects (it is possible to set up a lot of hybridization reactions and then wash/elute them).



• To reuse Dynabeads for the same RNA sample just wash them in BB.



• Do not heat magnetic concentrator for more then 70°C! Otherwise it looses magnetic properties.