MRNA purification on Dynabeads

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Содержание


NB! pre-PCR operation
NB! Auto-calculation works only in Internet Explorer.



[править] polyA RNA purification

  • check, that you have:
» rotator at NT
» magnetic stand
» 70°C water bath
» 1.7 and 0.5ml LoBind tubes



  1. calculate the binding mix recipies for all samples:

    number of samples:
    RNAAddDynabeadsW-buffer
    Noquantityvolume
    1
    2
    3
    4
    5
    6
    7
    8
    9
    10
    11
    12
    13
    14
    15
    16
    17
    18
    19
    20
    total: 


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

  2. prepare Dynabeads:
    • take Dynabeads for all samples (Vµl):
    • repeat two times:
      ○○   vortex beads 30 sec;
      ○○   magnetic rack 1 min and remove supernatant;
      ○○   add Vµl of B-buffer:

  3. prepare total RNA:
    • for each RNA sample reserve 1.5ml Sup-tube for collection of all washes;
    • transfer RNA into LoBind tube;
    • place RNA at 65°C for 2 min, then - immediately on ice;
    • if necessary (see auto-calculation table), add "10mM Tris-HCl" or "B-buffer"

  4. bind RNA to Dynabeads:
    • combine Dynabeads and RNA, mix carefully;
    • rotate 30-45 min at NT (~10rpm);

  5. wash Dynabeads:
    • magnetic rack 1 min;
    • transfer supernatant into prepared Sup-tube;
    • 2x wash in W-buffer:
    ○○   resuspend beads in W-buffer;
    ○○   vortex beads, magnetic rack 1 min and transfer supernatant into Sup-tube;
  6. (optional) second mRNA purification on Dynabeads:
    • resuspend beads in 50µl of 10mM Tris-HCl;
    • put for 2 min at 70°C, then immediately on ice;
    • add 50µl of B-buffer and mix carefully;
    • repeat "binding RNA to Dynabeads" and "washing Dynabeads";


  7. preheat special(!) magnetic concentator;


  8. two times mRNA elution (in two tubes):
    ○○   resuspend in 10µl of 10mM Tris-HCl;
    ○○   put for 2 min at 70°C;
    ○○   preheated magnetic rack 1 min and collect supernatant;
    • measure concentrations of both eluates;
    • snap freeze eluates and Sup-tube in liquid nitrogen, store at -20°C;

  9. (optional, for regeneration of beads) collect Dynabeads:
    • resuspend beads in original volume of S-buffer;
    • transfer into "Oligo(dT) REGENERATION" stock tube



[править] Solutions

prepare solutions using Ambion's buffers


[править] B-buffer

Binding Buffer, store at +4°C

using LiCl Precipitation solution from Ambion Cat#AM9480: LiCl 7.5M, EDTA 50mM

conc.5ml10ml
Tris-HCl 1M, pH7.520mM100µl200µl
LiCl 7.5M, EDTA 50mMLiCl 1M, EDTA 6.7mM670µl1.33ml
H2OmQ4.23ml8.47ml

using home-made LiCl 7M

conc.stock5ml10ml
Tris-HCl, pH7.520mM1M100µl200µl
LiCl1M7M714µl1.429ml
EDTA2mM0.5M20µl40µl
H2O mQ4.17ml8.33ml



[править] W-buffer

Washing Buffer, store at +4°C

using LiCl Precipitation solution from Ambion Cat#AM9480: LiCl 7.5M, EDTA 50mM

conc.5ml10ml
Tris-HCl 1M, pH7.510mM50µl100µl
LiCl 7.5M, EDTA 50mMLiCl 0.15M, EDTA 1mM100µl200µl
H2OmQ4.85ml9.7ml

using home-made LiCl 7M

conc.stock5ml10ml
Tris-HCl, pH7.510mM1M50µl100µl
LiCl0.15M7M107µl214µl
EDTA1mM0.5M10µl20µl
H2O mQ4.83ml9.67ml



[править] Tris-HCl, 10mM pH7.5

store at +4°C

conc.stock5ml10ml
Tris-HCl, pH7.510mM1M50µl100µl
H2O mQ4.95ml9.9ml



[править] S-buffer

Storage Buffer, store at +4°C

conc.stock5ml10ml
Tris-HCl, pH7.5250mM1M1.25ml2.5ml
EDTA20mM0.5M200µl400µl
Tween2010%0.1%50µl100µl
Sodium azide10%0.02%10µl20µl
H2O mQ3.49ml6.98ml




[править] Notes

  • Total time of the protocol is ~2h (~3h for double mRNA purification). Protocol should not be interrupted untill elution. 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
» not to dry out the beads
» RNase free work
» do not heat magnetic concentrator to more than 70°C! Otherwise it looses magnetic properties.


  • Binding mix recipe
» 0.5µl of Dynabeads suspension per 1µg of RNA (minimum 50µl, for precious samples - 100µl)
» optimal hybridization conditions are obtained if VRNA = VDynabeads:
if RNA volume is smaller than volume of Dynabeads suspension, — fill it up with Tris-HCl, 10mM pH7.5
if RNA volume is larger, — add B-buffer: volume = VRNA - VDynabeads


Dynabeads Oligo(dT)25

concentration5mg/ml
diameter2.8µm ± 0.2µm
surface area3-7m2/g
density~1.6g/cm3
magnetic mass susceptibility120 ± 25 x 10-6 m3/kg
purification cost7.6€ for 50µl of Dynabeads Oligo(dT)25


Double mRNA purification

  • After one round of purification ~50% of eluate is ribosomal RNA (independent on washing quality). This riboRNA is specifically bound to mRNA. To decrease ribosomal RNA content it is necessary to repeat the purification.
  • Intact ribosomal RNA is a much worse substrate for primer extension reaction if compared with sheared one. So, for RNA-Seq library preparation we recomend one round of Dynabeads purification if ds cDNA will be synthesized from full-length mRNA, and double purification if mRNA is fragmented prior to ds cDNA synthesis.


Reusing of Dynabeads

  • To reuse Dynabeads for the same RNA sample just wash them in B-buffer.
  • It is necessary to regenerate beads, before using them for purification of another RNA sample. NB! regeneration of oligo-dT celullose gives strong background during RNA-Seq library preparation. So, think carefully before reusing Dynabeads for different RNA samples.



Open questions

  • this protocol uses lower amount of Dynabeads and longer hybridisation time if compared with a "comercial manual". For single purification it is works fine. It worth to check purification quality for double purification scheme. In principle, longer hybridization may lead to higher reassociation rate of mRNA and ribosomal RNA.


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