Biology Reference
In-Depth Information
activation is made by the
Pac
I restriction endonuclease (for
most Ad5-derived constructs including the here described
pBA5-FRT-derived constructs,
see
Fig.
2b
) in a 100
μ
L stan-
dard reaction.
3. Add 50
L phenol/chloroform and vortex for 20 s.
4. Spin tubes in a microcentrifuge at maximum speed for 5 min at
room temperature.
5. Transfer 90
μ
μ
L of the aqueous upper phase into a fresh tube.
6. Add 10
L 100 % etha-
nol. Mix with fl icking by fi nger tips. The precipitated DNA
should become visible immediately. Incubate for 5 min at
room temperature.
7. Spin down tubes in a microcentrifuge at maximum speed for
10 min at room temperature.
8. Remove supernatant completely and immediately dissolve pel-
let in 20
μ
L 3 M Na-acetate (pH 4.5) and 200
μ
L sterile deionized water.
9. The day before transfection plate 2.5 × 10
5
/well 293 cells to a
6-well plate (
see
Note 22
).
10. Transfect 293 cells with 6-8
μ
g) activated Ad-BAC
DNA using any commercial transfection reagent (optimized
for 293 cells) according to the manufacturer's instructions.
Incubate the transfected plates for 3 days under standard cell
culture conditions.
11. On the second day after transfection plate out 2.5 × 10
5
/well
293 cells to a 6-well plate (
see
Note 22
).
12. On the third day after transfection (
see
Note 23
) harvest cells
by rinsing the plate with the medium several times until all cells
are detached from the plate surface. Usually there is no need to
use cell scraper, but make sure that you remove all cells from
the well.
13. Collect cell suspension in a sterile 15 mL Falcon tube by cen-
trifugation for 5 min at 200 ×
g
.
14. Remove supernatant and resuspend cell pellet in 400
μ
L (ca. 2-3
μ
μ
L
DMEM + 10 % FCS.
15. Place tube into liquid nitrogen for 3 min.
16. Immediately place tube in 37 °C water bath until cells have
thawed completely.
17. Repeat
steps 15
and
16
twice.
18. Centrifuge sample for 10 min at 3,500 ×
g
at room temperature
and transfer supernatant (lysate) into a fresh 1.5 mL Eppendorf
tube.
19. Infect one or two wells of the plate from
step 11
with 200
L
of lysate for each well. Incubate plates for 3-4 days under
μ
