mRNA Purification
I. Prepare oligo-dT cellulose
- use40 mgoligo-dT cellulose /1 mgtotal RNA
- swell oligo dT-cellulose in elution buffer
- wash oligo dT-cellulose4 xwith elution buffer (30 sec. full speed spin in between)
- equilibrate oligo-dT cellulose with2 to 3washing steps using 1x binding buffer
II. mRNA purification
- bring1 mgtotal RNA with H 2 O to600 µl
- incubate 4 min at65 °C
- add600 µl2x binding buffer
- add to40 mg1x binding buffer equilibrated oligo-dT cellulose
- incubate15 min at RTon a rolling incubator or vortex several times in between
- spin oligo-dT cellulose down, discard supernatant
- wash2 xwith1x binding buffer
- wash2 xwithwash buffer
- elute with250 µl elution buffer at 37 °C for 5 min
- spin and keep supernatant (for second round of purification or precipitation)
- elute oligo-dT cellulose again with250 µl elution buffer
- spin and keep supernatant (for second round of purification or precipitation)
- combine eluate and addH2 O to 600 µl
- repeat mRNA purification by starting again with 4 min incubation at 65 °C
III. Recover mRNA
- add50 µl 4 M NaCland precipitate with2 vol. cold EtOH
- incubate 1 h at -20 °C
- spin 10 min full speed, wash with 70 % EtOH, air dry and dissolve in20 µl H2 O
- read A260 of1 µl(100 to 250 fold diluted)
- run1 µg( and 10 µg total RNA as comparison) on a 1.2 % agarose Formaldehyde gelrecovery = 1-2 % of the total RNA (10-20 µg mRNA / 2 mg total RNA)
Buffers:[all buffers should be made with DEPC treated/autoclaved components!] oligo dT cellulose (type7, Pharmacia)2 x binding buffer:1 M NaCl; 20 mM Tris pH 7.5; 2 mM EDTA; 0.1 % SDS[50 ml: 12.5 ml 4 M NaCl; 1 ml 1 M Tris; 200 µl 0.5 M EDTA; 500 µl 10 % SDS] 1 x binding buffer:0.5 M NaCl; 10 mM Tris pH 7.5; 1 mM EDTA; 0.05 % SDS[50 ml:6.25 ml 4 M NaCl; 500 µl 1 M Tris; 100 µl 0.5 M EDTA; 250 µl 10 % SDS] wash buffer:0.2 M NaCl; 10 mM Tris pH 7.5; 1 mM EDTA; 0.05 % SDS[50 ml:2.5 ml 4 M NaCl; 500 µl 1 M Tris; 100 µl 0.5 M EDTA; 250 µl 10 % SDS] elution buffer:10 mM Tris pH 7.5; 1 mM EDTA; 0.05 % SDS[50 ml:500 µl 1 M Tris; 100 µl 0.5 M EDTA; 250 µl 10 % SDS]
