Biology Reference
In-Depth Information
10. Centrifuge the cells for 5 min at 7,000 ×
g
on +1 °C. Pour off
all the supernatant and without inverting the tube blot out the
remaining fl uid by a Kleenex tissue from the upper 2/3 of the
wall of the tube. Put the bacterial pellet back on ice for 1-2 min.
Resuspend the bacterial pellet by pipetting up and down in the
remaining supernatant and check the volume of the suspen-
sion. It should be in between 120 and 150
μ
L. Add ~600
μ
L
L.
11. Aliquot a 35 µL electrocompetent cell suspension into 0.5 mL
Eppendorf tubes and snap-freeze them dropping the tubes
directly into liquid nitrogen. Store the frozen vials on −80 °C.
Alternatively, keep on ice the aliquots, that will be immediately
used.
12. For each expression construct thaw up or reserve one vial of
pBA5-FRTxpCP20 electrocompetent cells on ice together
with one vial for the control.
13. Transfer one aliquot of the electrocompetent cells and ~100 ng
of the pO6A5-CMV construct (
step 1
) to 0.2 mm electro-
poration cuvette on ice and mix carefully. Add sterile deionized
water instead of the donor plasmid into the control cuvette.
14. Electroporate mixes with 2,500 V, 200 W, and 25
of ice-cold sterile 10 % glycerol to get a fi nal volume of 700
μ
F in Gene
Pulser (or equivalent electroporation equipment). When
accomplished, mix bacteria with 1 mL LB without any antibi-
otic and transfer them into 1.5 mL Eppendorf tubes. Incubate
at 43 °C for 1 h in a shaking incubator.
15. Plate out 10 and 100
μ
μ
L of the bacterial suspension on LB
g/mL
kanamycin. Incubate the cultures overnight at 43 °C (
see
Note
20
).
16. Inoculate 2-3 fl asks with 100 mL LB medium supplied with
25
plates containing 25
μ
g/mL chloramphenicol and 25
μ
g/mL kanamycin with a
single colony from the plate incubated at 43 °C and grow cul-
ture overnight at 37 °C with shaking (
see
Note 21
).
17. Prepare BAC DNA using an appropriate purifi cation kit
according to the manufacturer's instructions (
see
Note 9
).
Verify the constructs by restriction analysis and sequencing.
μ
g/mL chloramphenicol and 25
μ
1. Prepare 10-30
g BAC DNA from the desired recombinant
using an appropriate purifi cation kit according to the manufac-
turer's instructions (
see
Note 9
). It is recommended to rescue
two independent verifi ed clones for the same construct to
ensure consistent data in upstream experiments.
2. For virus reconstitution activate 10
μ
3.4 Reconstitution
of Recombinant
Adenoviruses
from BACs
g of Ad-BAC DNA by
linearization with the appropriate restriction endonuclease,
cutting Ad-BACs at specifi c ITR-fl anking sites. Usually this
μ
