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Mullins Lab : Protocols : mRNA Isolation
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University of Washington School of Medicine, Department of Microbiology     

Selection of Poly A+ RNA (Katze Lab w/modifications)

Reagents:

  • Oligo (dT)-cellulose Type 7 (Pharmacia)

  • Polypropylene chromatography columns (BioRad)

  • 0.1 N NaOH/5 mM EDTA

  • 2x Column Loading Buffer (40 mM Tris pH 7.5, 1 M LiCl, 2 mM EDTA, 0.2% SDS)

  • dH20

  • 4 M LiCl

  • Linear acrylamide (5 mg/ml)

  • 100% EtOH

 

Protocol:

  1. Pour a small amount of Oligo (dT)-cellulose powder into a conical tube and suspend in 1X Column Loading Buffer.

  2. Transfer into the column that is sitting in a 15ml conical tube (up to 10mg RNA can be selected per ml of oligo (dT). Oligo (dT)-cellulose packed to 0.4ml for 1mg total RNA gave very good yields. Expect ~15-50 µg poly A+ RNA per mg of total RNA.

  3. Wash with 10 volumes each

    1. sterile dH2O

    2. 0.1N NaOH/5mM EDTA

    3. sterile dH2O

  4. Wash with 5 volumes 0.1x Column Loading Buffer.

  5. Wash with 10 volumes dH2O.

  6. With pH paper, check to make sure the pH of the column effluent is less than 8.

  7. Wash the column with 1x Column Loading Buffer, then replace the catch tube with a new one.

  8. In the meantime, prepare the RNA sample:

    1. dissolve in 500 ml dH2O

    2. heat at 65oC for 5 minutes

    3. add an equal volume of 65oC 2x Column Loading Buffer

    4. let cool to RT on benchtop

  9. Apply RNA sample to column.

  10. Collect the flow through, heat again to 65oC for 5 minutes, allow to cool to almost RT and reapply to column.

  11. Collect the effluent; this is the poly A- fraction.

  12. Replace the catch tube and wash with 10 volumes of 1x Column Loading Buffer.

  13. Replace the catch tube again and elute the poly A+ RNA with 2 ml 65oC dH2O.

  14. Spin at 2000 rpm, 4oC for 10 minutes to remove contaminating gel.

  15. Read O.D. 260/280 and calculate concentration.

  16. Ethanol precipitate with

    1. LiCl added to 0.2M (1:4)

    2. 100 µg linear acrylamide (carrier)

    3. 2.5 volumes 95% EtOH.

    Always do the precipitation overnight.

  17. Regenerate the column by sequential washing with 10 volumes each of 0.1 N NaOH/5 mM EDTA, dH2O and 1x Column Loading Buffer. Columns may be stored at -20 °C and used up to 3 times, depending on the amount of RNA selected.

 

Note: If you must break the 2 ml into aliquots for eppendorf tubes, use 20 µg linear acrylamide per aliquot.

 

 

 

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

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jl, 19may02

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