1) Split 293T cells out the day before transfection. 2-2.5 X 106 cells/6cm plate generally works well. At the time of transfection cells should be 70-85% confluent in 3mL of DMEM w/10%FCS.
2) Add 2ul of 50mM Choloroquine to plates prior to transfection.
3) To a 15mL tube, add 5ug structural
vector (e.g. CPR/\Env), 3ug transfer
vector (e.g. pFLX-5CL), and 2ug envelope vector (e.g. pCI-VSVG).
4) Add 61ul of 2M CaCl2(Malinkrodt
only) - try to wash the DNA down the
5) Add 430ul sterile ddH2O - must be room temperature.
6) Spin liquid to bottom of tube.
7) Add 500ul of 2XHBS and bubble
2-10sec in the water/CaCl/DNA
mix. For information on making 2XHBS see the Phoenix protocol page.
8) Add the mixture dropwise to the plated cells.
9) Incubate cells at 37 degrees for 8hrs.
10) Change media to fresh DMEM w/10% FCS.
11) Change media again in 22hours.
12) Collect supernatant at 48hrs
post-transfection. (NB: supernatants can be
collected as early as 35 hours post-transfection and as late as 72 hours and
will still contain good titre - so you can get 2 or 3 virus collections per
transfection, peek is somewhere between 48 and 60 hours post. Also,
moving plates to 32 degrees ~8hrs prior to collection can increase yields.)
13) Clear the supernatant by spinning
in a 15mL tube at 1500RPM for 5
minutes and/or by filtering through a .45um filter. (if you are filtering you
will lose some titre, this loss can be minimized by pre-wetting the filter
with serum or serum-containing media and by using low-protein binding
14) OPTIONAL: Virus can be concentrated
by centrifugation at >40000XG
for 1.5-2hrs. I use 50mL tubes in a fixed angle Beckman JA.20 or
JA25.50 at 20000 RPM. Conventionally you concentrate in an
ultracentifuge (SW41 or SW28 rotor) at 50000XG for 1.5hrs. The pellet
should be resuspened in TNE (Tris-NaCl-EDTA).
15) Target cells are infected by
adding virus-containing supernatant or
purified, concentrated virus diluted in media in the presence of polybrene
(1000X stock is 5mg/mL) or protamine sulfate (1000X = 8mg/mL). For
best results cells should be infected in multi-well plates and then spun at
2500RPM for 90 minutes at 30degress immediately following addition of
virus. Spin infection significantly enhances infectivity.
16) Place cells containing virus
at 32 degrees for 6-36hrs - usual overnight
17) Change infected cells to fresh media.
18) Analyze protein expression >48hr post-infection.
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