SV40 in vivo Replication Assay
Replication assays are described as modifications of published procedures (Hertz, 1986;
Weichselbraun, 1989)
1. SV40 replication assays are best done in CV1 or CV1-P African green monkey
kidney cells. However, these assays can be done in human cells that are semipermissive for replication.
2. Split cells the day before (3x105) on to p60 dishes so they 30-40% confluent at the
day of transfection. Typically, we transfect 2.5 µg pSVori (a plasmid containing
the SV40 origin of replication, pSV01EP) + 2.5 µg pCMV LT (a LT expression
plasmid that does not contain an origin) using the BES calcium phosphate
method. If you add in another plasmid be sure that it doesn’t have an origin of
replication and also that the total amount of DNA is adjusted to 5 µg e.g. with
pUC. Remember to include a negative control: No LT.
3. At 48 hrs post transfection harvest the cells. Wash 2x with PBS. Remove as much
PBS as you can by tilting the dishes. Lyse in 600 µl TE + 0.6 % SDS for 10 min
at room temperature. After gently scraping the dish with a cell scraper, the
viscous lysate is poured into a 1.5 ml microcentrifuge tube. 200 µl is removed for
later Western analysis of LT expression. Add 100 µl of 5M NaCl to the remaining
400 µl of extract and mix by inverting gently 20 times. The same cell scraper can
be used for multiple samples if you rinse it between samples. Incubate overnight
(or longer) at 4 ˚C.
4. Spin the samples in a microcentrifuge 14,000 rpm at 4˚C for 30 min. Remove the
supernatant carefully and transfer no a new tube (avoid white pellet).
5. Extract the supernatant 3 times in the fume hood with an equal volume of phenol/
chloroform (constituting lower phase in tube, since it’s heavier). Each time
vortex, and then spin down 5 min at 14,000 rpm. Transfer supernatant to a new
tube and extract again. Each time the lower phase is discarded as chemical waste.
Finally, ethanol precipitate by adding 1/10th volume (50 µl) 3M NaAc pH 5.5 and
two volumes (1 ml) of ethanol. Spin 15 min 14,000 rpm at 4 ˚C.
6. Carefully aspirate avoiding the pellet (don’t let the samples sit after the centrifuge
has come down, proceed immediately, the pellet might come lose). Wash pellet
with 500 µl ice cold 70% ethanol. Spin again 2 min. Aspirate with a fine tip
VERY carefully to avoid the pellet. Air dry 10 min.
7. Resuspend sample in 50 µl sterile water. Half of the sample is then digested; store
the remaining at -20 ˚C. Set up an overnight digest in a 50 µl total volume. Use a
minimum of 3 units of HinCII and 3 units of DpnI together with NEB Buffer 4.
Incubate overnight in the 37 ˚C room. If you want to, you can set up control
digests of pSVori with HincII/ DpnI isolated from either Dam+ or Dam- bacterial
hosts. These are excellent markers in the Southern blot that follows.
8. After digestion with HinCII/ DpnI the fragments from approximately 1/10th of a
p60 dish is typically examined by Southern. E.g. take 10 µl of digest, add
appropriate sample dye and load on a 1.5% agarose in ½ x TBE gel. Include
ethidium bromide in the gel. As size markers, run 50-100 pg of pSVori control
digest.
9. Look at the gel on the UV transilluminator. You should see a smear of bands,
uniform between the samples.
10. Prepare probe for Southern blotting. Digest 10 µg of pSVori with EcoRI and
isolate the 314 bp fragment encompassing the origin. Gel-purify using Qiaquick
gel purification kit from Qiagen. Prepare probe using the random-primed DNA
labeling kit from Roche (cat no 11004760001). Purify the probe afterwards using
a Bio-Rad Bio-Spin 30 chromatography column or similar. This gets rid of
unincorporated nucleotides. The specific activity of the probe should be >108
cpm/µg (count in a scintillation counter).
11. Southern blotting protocol:
12. Trim the gel so you only have the area you’re interested in. Soak the gel for 30
min in denaturation solution (1.5M NaCl, 0.5M NaOH) on a shaker at room
temperature.
13. Replace buffer with neutralization solution (3M sodium acetate, pH 5.5) and
shake for 30 min more.
14. Set up the Southern transfer. Place a glass plate across a clean Pyrex dish
containing about 1” of 20xSSC. Place a wick (2 sheets of Schleicher and Schuell
GB002 blotting paper) across the glass plate connecting on both sides with the
20xSSC. Place 4 sheets of GB002 paper cut exactly the size (or slightly larger) of
the gel in the middle of the wick. Place the agarose gel (wells facing down) on top
of the 4 sheets. Cut a piece of Immobilon Ny+ membrane (Millipore) same
dimensions as the gel. Wet it first in 100% methanol, then in 20xSSC. Place it on
top of the gel (don’t move it around) and press it down. Use a 10 ml pipette to
make sure there are no air bubbles. Place another 4 sheets of GB002 paper on top
of the membrane. Cover carefully the whole assembly with Sealwrap. Cut
carefully with a razorblade the Sealwrap around the gel and remove it. Stack
paper towels on top of the GB002 paper. They have to be cut into the dimensions
of the gel. Put at least paper towels to a height of 1”. Finish it off by placing a
glass plate on top of the towels and then a weight.
15. Remove the membrane and place it into a folded Whatman filter paper. Mark the
gel side with a pencil. Either bake the membrane at 80˚C for 1 hr or UV crosslink
it in a Stratalinker (gel side facing up).
16. Pre-hybridization. Seal the membrane in a seal-a-meal bag or roll it up in a
hybridization flask using 10 ml of hybridization solution (50% formamide,
5xSSC, 50mM sodium phosphate, pH 6.5, 5x Denhardt’s, 250 µg/ml of Salmon
sperm DNA (boiled and then frozen, to denature), 0.1. % SDS). Incubate 1 hr at
42 ˚C.
17. Boil the probe for 5 min to denature the DNA, then cool on ice.
18. Hybridization. Remove the pre-hybridization solution and replace with fresh
hybridization solution containing the probe (did you boil it <1 hr before?).
Incubate at 42 ˚C (shaking or rotating depending on the setup) overnight.
19. Remove the hybridization solution and dispose of as radioactive waste. Rinse in
1st wash buffer (2x SSC, 0.1 % SDS). Wash 4 times for 5 min each in 1st wash
buffer on a shaker at room temperature.
20. Wash twice for 15 min (can go longer time) each in 2nd wash buffer (0.1xSSC,
0.1% SDS) at 50 ˚C.
21. Blot membrane briefly on filter paper then proceed with autoradiography.
Solutions:
20xSSC: 3M NaCl,0.3M Na citrate pH 7
Formamide (Invitrogen), store at -20˚C
50x Denhardt’s: 1% Ficoll type 400, 1% polyvinylpyrrolidone, 1% bovine serum
albumin. Store at -20˚C.
0.5M Na phosphate pH 6.5
Denaturation solution: 60 ml 5M NaCl, 8 ml 12.5M NaOH, water up to 200 ml
Neutralization solution: 8.2 g anhydrous sodium acetate, 70 ml of water. Adjust pH to 5.5
then volume to 100 ml.
Hybridization mix:
Final conc.
50% formamide
5x SSC
50 mM Na
phosphate pH 6.5
5x Denhardt’s
250µg/ml Salmon
sperm DNA
0.1% SDS
Stock
100%
20x
0.5M
Amount for 10 ml
5 ml
2.5 ml
1 ml
Amount for 20 ml
10 ml
5 ml
2 ml
50x
10 mg/ml
1 ml
0.25 ml
2 ml
0.5 ml
10%
0.1 ml
0.2 ml
1st wash buffer:
Final conc.
2xSSC
0.1% SDS
Water
Stock
20xSSC
10%SDS
Amount for 2 l
200 ml
20 ml
1.78 l
2nd wash buffer:
Final conc.
0.1xSSC
0.1% SDS
Water
Stock
20xSSC
10%SDS
Amount for 1 l
5 ml
10 ml
1.985 l