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Joe's mRNA prep
Joe's mRNA prep
(Audrey Gasch, Pat Brown [modified by S.M. Hettenbach])

Oligo-dT cellulose prep

  1. Dump Ambion vial contents into 50 ml c/f tube
  2. Add 10 ml 1x NETS to vial, cap, rinse vial, dump 10 ml into c/f tube
  3. Spin tube 3000 rpm, 2 min at 4°C in swinging bucket with no brake, remove s/n
  4. Add 10 ml 1x NETS to tube, resuspend contents by swirling
  5. Spin tube 3000 rpm, 2 min at 4°C in swinging bucket with no brake, remove s/n
  6. Repeat steps 4 and 5 two more times for a total of 4 washes
  7. Add 10 ml 2x NETS and store at 4°C
  8. Prep cellulose the day before you need it

mRNA prep

  1. Dilute 1 mg total RNA to 1 ml with 10mM Tris pH7.4 in c/f tube
  2. In a 2 ml BioRad mini column(#731-1550) sealed on bottom, mix 1 ml resuspended resin with 1 ml of 1 mg/ml total RNA from step 1. Cap and parafilm column, tape to rocker, and bind at RT for 1 hour on 5.5 setting
  3. Break bottom off column and place in 16 x125 mm test tube and allow contents to flow through
  4. Wash resin five times with 0.7 ml 1x NETS, slow flow is normal
  5. Transfer column to support and elute 2x with 0.7 ml 10 mM Tris pH 7.4 preheated to 70°C into separate 1.7 ml tubes. Elute with 150 l of the 0.7 ml volume at a time keeping rest at 70°C.
  6. Add 0.7 ml chloroform to each tube to remove residue cellulose, mix well, spin 13,000 rpm for 1 min at RT, transfer upper phase to new tube
  7. Precipitate with 0.1 volume 3M NaOAc pH 7.0 and 1 vol isopropanol at -20°C for 1 hour
  8. Spin 13,000 rpm for 30 min at 4°C
  9. Remove s/n and air dry
  10. Resuspend both tubes in final volume of 20l of DEPC ddH2O
  11. Spec OD260 & OD280 on 1 l mRNA. 1 OD260 is equal to 40 g/ml of RNA
  12. Store at -80°C, avoid repeat freezing and thawing