Biology Reference
In-Depth Information
3.3.3 Third HC-Ad Vector
Amplifi cation
1. Release vector particles from the cells by three repeated
freeze-thaw cycles in liquid nitrogen and a water bath (37 °C).
Gently swirl the tubes with the cell suspensions while freezing
and thawing.
2. Replace the medium of one 150-mm dish with 90 % confl uent
116 cells seeded 2 days before with 15 mL of fresh, supple-
mented medium.
3. Infect cells with 0.5 mL of the freeze-thaw lysate obtained in
step 1 . Coinfect the cells with 15-20 MOI of helper virus.
One hour after infection fi ll up to 30 mL with supplemented
medium.
4. 48 h after infection the cells should show a complete cyto-
pathic effect (CPE) characterized by cell rounding and detach-
ment from the dish. Harvest the cells in their medium by
pipetting up and down or scraping.
5. Spin down cells at 400 × g for 10 min. Aspirate and discard
supernatant. Resuspend the cells in 2 mL PBS. Prepare
genomic DNA from a 200
L aliquot of the cell suspension
using the QiaAmp DNA Mini Kit (Qiagen, Hilden, Germany)
according to the manufacturer's instructions ( see Note 12 ).
Snap-freeze the remaining cell suspension (1.8 mL) in liquid
nitrogen and store at −80 °C until further use.
μ
1. Release vector particles from the cells by three repeated
freeze-thaw cycles in liquid nitrogen and a water bath (37 °C).
Gently swirl the tubes with the cell suspensions while freezing
and thawing.
2. Replace the medium of two 150-mm dishes (two dishes per
vector) with 90 % confl uent 116 cells seeded 2 days before
each with 15 mL of fresh, supplemented medium.
3. Infect each dish with 0.5 mL of the freeze-thaw lysate obtained
in step 1 . Coinfect the cells with 15-20 MOI of helper virus.
One hour after infection fi ll up to 30 mL (per dish) with sup-
plemented medium.
4. 48 h after infection the cells should show a complete cyto-
pathic effect (CPE) characterized by cell rounding and detach-
ment from the dish. Harvest the cells in their medium by
pipetting up and down or scraping.
5. Spin down cells at 400 × g for 10 min. Aspirate and discard
supernatant. Combine and resuspend the cells from both
dishes in 4 mL PBS. Prepare genomic DNA from a 200
3.3.4 Fourth Vector
Amplifi cation
L
aliquot of the cell suspension using the QIAamp DNA Mini
Kit according to the manufacturer's instructions. Snap-freeze
the remaining cell suspension (3.8 mL) in liquid nitrogen and
store at −80 °C until further use.
μ
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