Transient transfection:

Day 1 (preferably early):
For each transfection trypsinize one confluent 10-cm plate of cells and plate everything onto one 15-cm plate (for multiple plates, mix cells after trypsinizing and plate from the mixture to get more homogenous plates).

Day 2:
When plates are 50-80% confluent transfect the cells using Lipofectamine as described by manufacturer.
For each 15-cm plates use:
100 µl Lipofectamine; mix with 1.4 ml O-MEM
25 µg plasmid; mix with 1.5 ml O-MEM
Mix Lipofectamine and plasmid mixtures.
Leave at RT 15-45 min.
Add 12 ml O-MEM.
Remove medium from cells
Wash with 20 ml O-MEM.
Add Lipofectamine/plasmid mixture (15 ml total) to cells.
Incubate 5-24 hours in incubator
Remove transfection mixture.
Add DMEM/10% FBS.
Incubate 24-48 h.

Immunoprecipitation:
Day 4:
Optional: Add cycloheximide to 50 µg/ml to cells. Incubate 2 h.
Wash cells with PBS.
Add 10 ml PBS and scrape off cells using a rubber policeman.
Transfer to 14 ml Falcon tube.
Spin 1,000 rpm, 10 min.
Remove PBS.
Resuspend in ice-cold 400 µl hypotonic gentle lysis buffer and transfer to eppendorf tube.
Incubate on ice, 10 min.
Add NaCl to 150 mM. Incubate on ice 5 min.
Spin 14,000 rpm, 4C, 15 min.
Transfer µl supernatant to 1 ml of Trizol. (Total extract control). Leave at RT, 5 min. Store at -80C.
Transfer remainder of supernatant to tube containing 100 µl anti-FLAG-M2 agarose slurry, which has already been washed twice with 1 ml NET-2 (1,000 rpm, 1 min for each wash).
Nutate at 4C, ≥4h.
Wash beads 8 times with 1 ml NET-2 (ice-cold). Spin 1,000 rpm, 1 min for each wash. (Try to remove as much wash buffer as possible without removing beads in each wash).
After last wash, add 200 µl NET-2 containing 1 mg/ml FLAG peptide, 40 units RNasin.
Genly shake tubes at 4C for 2 h.
Spin 1,000 rpm, 1 min.
Transfer 200 µl supertant to 1 ml Trizol. Leave at RT, 5 min. Store at -80C.

RNA preparation:
Day 5:
Prepare RNA according to manufacturer:
Leave Trizol mixture at RT, 5 min.
Add 200 µl Chloroform.
Mix by shaking for 30 sec or more.
Leave at RT, 10 min.
Spin 14,000 rpm, 4C, 15 min.
Transfer supernatant to new tube. Avoid interphase!!
Add 10-20 µg of glycogen.
Add 0.7 volumes of isopropanol. Mix and leave at RT, 10 min.
Spin 10,000 rpm (not faster!), 4C, 30 min.
Remove supernatant carefully.
Wash with 70% EtOH.
Dry.
Dissolve in 20 µl formamide load buffer.
Northern blotting:

Day 6:
Gel preparation:
Run in hood with MOPS running buffer at 120 V for 1 h. followed by 160 V for 4-6 h. (Check pH at anode and catode from time to time).
Transfer gel (in tray) to 10xSSC. Wash for 30-60 mins.
Set up capilary gel transfer over night.

Day 7:
UV X-link membrane in Stratalinker.
Wash briefly with water.
Prehybridize membrane with 20 ml hybridization buffer for 2 h. at 50C.
Add 1-5x106 cpm of probe to 0.8 ml hybridization buffer and incubate at 95C, 1-2 mins.
Add everything to tube with prehybridized membrane.
Hybridize over night at 50C.

Day 8:
Wash two times 15 min at room temperature with 0.5xSSC, 0.1% SDS.
Wash one time 15 min at room temperature with 0.1xSSC, 0.1% SDS.
Wash one time 15 min at 60C with 0.1xSSC, 0.1% SDS.
Wash briefly with water.
Wrap in Saram Wrap and expose to film.

Buffers:
Hypotonic gentle lysis buffer:
10 mM Tris-HCl pH 7.5
10 mM NaCl
2 mM EDTA
0.5% Triton-X100
added freshly:
1 mM PMSF
1 µM aprotinin
1 µM leupeptin

NET-2:
50 mM Tris-HCl pH7.5
150 mM NaCl
0.05% Triton-X100

Formamide load buffer:
Deionized Formamide
5 mM EDTA
0.1% Bromophenol blue
0.1% Xylene Cyanol
2xMOPS/Formaldehyde load buffer:
MOPS running buffer: