我的参考步骤是invitrogen的，感觉和园子里写的不太一样，请大家帮我指点一下，万分感谢！
1. The day before transfection, trypsinize and count the 293A cells, plating them
at 5 x 105 cells per well in a 6-well plate. Plate cells in 2 ml of normal growth
medium containing serum.
2. On the day of transfection, remove the culture medium from the 293A cells
and replace with 1.5 ml of normal growth medium containing serum (or Opti-
MEM® I Medium containing serum). Do not include antibiotics.
3. Prepare DNA-Lipofectamine™ 2000 complexes for each transfection sample
by performing the following:
• Dilute 1 μg of Pac I-digested pAd-DEST expression plasmid DNA in
250 μl of Opti-MEM® I Medium without serum. Mix gently.
• Mix Lipofectamine™ 2000 gently before use, then dilute 3 μl in 250 μl of
Opti-MEM® I Medium without serum. Mix gently and incubate for
5 minutes at room temperature.
• After the 5 minute incubation, combine the diluted DNA with the diluted
Lipofectamine™ 2000. Mix gently.
• Incubate for 20 minutes at room temperature to allow the DNALipofectamine
™ 2000 complexes to form. The solution may appear cloudy,
but this will not impede the transfection.
4. Add the DNA-Lipofectamine™ 2000 complexes dropwise to each well. Mix
gently by rocking the plate back and forth. Incubate the cells overnight at
37°C in a CO2 incubator.
5. The next day, remove the medium containing the DNA-Lipofectamine™ 2000
complexes and replace with complete culture medium (i.e. D-MEM containing
10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin).
6. 48 hours post-transfection, trypsinize cells and transfer the contents of each
well to a sterile 10 cm tissue culture plate containing 10 ml of complete culture
medium.
Caution: Remember that you are working with infectious virus at this stage
and in all subsequent procedures. Follow the recommended guidelines for
working with BL-2 organisms (see page 5 for more information).
7. Replace culture medium with fresh, complete culture medium every 2-3 days
until visible regions of cytopathic effect (CPE) are observed (typically 7-10
days post-transfection). For an example, see the next page.
8. Replenish culture medium and allow infections to proceed until
approximately 80% CPE is observed (typically 10-13 days post-transfection).
9. Harvest adenovirus-containing cells by squirting cells off the plate with a
10 ml tissue culture pipette. Transfer cells and media to a sterile, 15 ml,
capped tube. Proceed to Preparing a Crude Viral Lysate作者: hot_hot_hot 时间: 2012-5-2 16:16
