This protocol is for four time-points in 12-well plates. Scale up or down as needed.

Day 1: Plate cells:

Split one or more 10-cm plate(s) with 80-100% confluent HeLa Tet-off cells to a final volume of 10-11 ml. Make sure to break up cell clusters to ensure single cells.

Dilute freshly split cell suspension 1:12 or 1:15 (depending on % confluency) in DMEM/10% FBS in a 50 ml conical in a total volume equal to or more than the number of wells to be plated (for e.g. make about 50 ml for 48 wells -12 experiments of 4 time-points).

Plate 1 ml per well in 12-well plates.

Incubate over night.

Day 2: Transfections

Using Mirus TransIT HeLaMonster (check manufacturers protocols if using other reagents)

Cells should be 40-60% confluent - not more.

For each of four wells:

1) Add a total of 4.5 μg plasmid to an eppendorf tube:

For B-globin reporters use:

45 ng pcBwtGAP3 UAC (internal control mRNA)

0.9 μg pPC or pcTET2 B-globin reporter

3.6 μg of other plasmid(s) - such as protein expressing or empty vectors

Optional: If using same reporters in all experiments, make a larger mix of internal control and B-globin reporter. For e.g., for 12 experiments, mix

45 ng x 12.5 of pcBwtGAP3 UAC (internal control mRNA)

0.9 μg x 12.5 of pPC or pcTET2 B-globin reporter

Then, divide the mix into 12.5 equal parts and pipet into separate eppendorfs.

2) Mix 225 μl O-MEM with 13.5 μl TransIT HeLa reagent

Add 0.225 μl of 1 mg/ml Tetracycline (in EtOH).

(a final concentration of 50 ng/ml Tet ensures no expression from the TET-driven promoter)

Incubate 5-20 mins at RT

3) Mix 5 μl HeLaMonster reagent with 45 μl ddw. Store on ice.

(Important: Thaw HeLaMonster reagent just before use, keep on ice and return to freezer immediately).

Optional: Bigger mixes can be made by multiplying O-MEM, TransIT reagent and Tetracycline volumes (Step 2) or HeLaMonster and ddw volumes (Step 3) by the number of experiments (+0.5).

4) Add 225 μl O-MEM/TransIT HeLa mix to the plasmid mix.

Incubate 5-20 mins at RT

5) To each 2.0-cm well of HeLa Tet-off cells:

Add 50 μl of O-MEM/TransIT/Plasmid mix.

6) To each 2.0-cm well of HeLa Tet-off cells:

Add 10 μl of diluted HeLaMonster reagent.

Place plates in incubator.

Day 4: Pulse-Chase assay.

1) Pulse transcription (Early morning):

Start transcription from Tet-promoter:

Wash wells carefully (!) with 1 ml PBS.

Add 1 ml DMEM/10% FBS.

Incubate ≈6 hours

2) Chase:

Stop transcription (the complete stop will take ≈20 mins):

To each well, add 10 μl of O-MEM/100 μg/ml Tetracycline

Place back in incubator.

3) Time points:

Take the first time point 20-30 mins after addition of tetracycline (t=0).

For each time point:

Wash cells with 1 ml PBS.

Add 0.5 ml Trizol. Pipet up and down until non-viscous.

Transfer to pre-labeled eppendorf tube.

Store in -20 C.

Day 5: RNA preps:

To each tube with 0.5 ml Trizol:

Add 100 μl Chloroform.

Mix by shaking for 30 sec or more.

Leave at RT, 10 min.

Spin 12,000 rpm, ≈10 min.

Transfer 250 μl supernatant to new tube. Avoid interphase!!

Add 0.7 volumes of isopropanol (175 μl). Mix and leave at RT, 10 min.

Spin 12,000 g (not faster!), 4C, 10 min.

Remove supernatant carefully.

Wash carefully with 0.5 ml of 70% EtOH. Spin at 7,500 g for 5 min, 4C.

Air dry.

Dissolve in 10 μl formamide load buffer.

Run 5 μl on a Northern Gel.