Joe's mRNA prep (Audrey Gasch, Pat Brown [modified by S.M. Hettenbach]) Oligo-dT cellulose prep - Dump Ambion vial contents into 50 ml c/f tube
- Add 10 ml 1x NETS to vial, cap, rinse vial, dump 10 ml into c/f tube
- Spin tube 3000 rpm, 2 min at 4°C in swinging bucket with no brake, remove s/n
- Add 10 ml 1x NETS to tube, resuspend contents by swirling
- Spin tube 3000 rpm, 2 min at 4°C in swinging bucket with no brake, remove s/n
- Repeat steps 4 and 5 two more times for a total of 4 washes
- Add 10 ml 2x NETS and store at 4°C
- Prep cellulose the day before you need it
mRNA prep - Dilute 1 mg total RNA to 1 ml with 10mM Tris pH7.4 in c/f tube
- 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
- Break bottom off column and place in 16 x125 mm test tube and allow contents to flow through
- Wash resin five times with 0.7 ml 1x NETS, slow flow is normal
- 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. - 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
- Precipitate with 0.1 volume 3M NaOAc pH 7.0 and 1 vol isopropanol at -20°C for 1 hour
- Spin 13,000 rpm for 30 min at 4°C
- Remove s/n and air dry
- Resuspend both tubes in final volume of 20
l of DEPC ddH2O - Spec OD260 & OD280 on 1
l mRNA. 1 OD260 is equal to 40 g/ml of RNA - Store at -80°C, avoid repeat freezing and thawing
|