• 1 day prior to transfection, plate tet-off cells at 30-50% density in 10cm plates (1 plate/sample).
• On the following day, transfect cells with 12ug of total plasmid using HeLa monster Rgt. (3.5ug mRNA reporter, 0.5ug of B-gap control reporter, other plasmid of interest, fill up rxn. with vector) (remember to transfect in the presence of tetracycline). Incubate cells O/N at 37 degrees C.
• 2 days later: in the morning, pulse transcription of reporter mRNA for 6-8 hrs by washing cells once in PBS and applying tet-free DMEM.
• Prior to harvesting lysates, incubate cells in a final conc. of 0.1ug/mL cycloheximide for 3 min at 37 degrees C.
• After 3 min, remove media and apply 10 mL of 1X PBS/0.1ug/mL cycloheximide. Put plate on ice.
• Wash cells twice with cold PBS/0.1ug/mL cycloheximide.
• Lyse cells on plate with 1mL of polysome extraction buffer (see below)
• Place extract in eppendorf tubes and incubate on ice for 10 min.
• Remove debris by spinning at 12,000xg for 10 min. Recover supernatant. These extracts are ready to be loaded on sucrose gradients of 10-50% consistency (keep samples on ice at all times).

To make sucrose gradients:

• Make 10, 20, 30, 40, and 50% sucrose solutions dissolved in polysome extraction buffer (-) Triton X-100
• Make a 10mL gradient by adding 2mL of each % solution (50% on bottom; 10% on top) to a ultra centrifuge tube

• Place parafilm tightly over each tube. Very, very slowly turn each gradient to its side (it may take 1-2 mins to turn tube without disrupting gradient). Lay tubes on their sides at 4 degrees C for 2 hrs. to allow for formation of a linear gradient.

• Add 800uL of extract to the top of each gradient and spin samples at 35,000 rpms at 4 degrees C for 190 min in an SW41 rotor (Have someone help you set this up the first time).

• When samples have finished spinning, using a fraction collector to collect 1mL fractions from the bottom of the tube to the top.
• To purify RNA, take 400uL of each fraction and add 600uL of guanidine HCL and 600uL of isopropanol. Vortex hard and incubate samples at –20 degrees C O/N.
• On the following day, centrifuge samples for 25 mins at 10,000 rpm.
• Wash pellet in 70% EtOH and resuspend in 400uL of TE.
• Reprecipitate samples by adding 40uL of NaOAC and 2.5 volumes of 100% EtOH. Let samples sit for >10 min. Spin at 10,000 rpm for 25 min.
• Wash pellets in 70% EtOH.
• Run samples on northern gel and blot as normal. Stain blots with methylene blue to observe ribosomal RNA bands (if desired). Probe blot as usual.

Polysome extraction buffer:

10mM Tris (7.4)
1% Triton X-100
15mM MgCl2
0.1ug/mL cycloheximide
0.3M NaCl
1mg/mL Heparin