Biology Reference
In-Depth Information
8. Resuspend the pellet in 1 mL M9 medium and plate out 1, 10,
and 100
L of the washed suspension from both the main and
the control reactions on DOG plates containing 25
μ
g/mL
chloramphenicol (
see
Note 14
). Incubate the plates at 32 °C
for 2-4 days.
9. Pick 10-20 single colonies and streak each one onto a quarter
of an LB plate containing 25
μ
μ
L chloramphenicol. Incubate
the plates at 32 °C overnight.
10. Pick single colonies from each quarter to inoculate 10 mL LB
containing 25
g/mL chloramphenicol and incubate the cul-
tures overnight at 32 °C.
11. Prepare BAC DNA by the small-scale protocol (
see
Note 9
)
using 9.8 mL of the overnight culture and subject them to a
restriction pattern analysis. Save the remaining BAC cultures at
+4 °C.
12. Streak a loop of the remaining cultures corresponding to two
colonies with correct restriction pattern onto MacConkey
plates (
see
Note 7
) supplemented with galactose and 25
μ
μ
g/
mL chloramphenicol.
13. Pick a well-isolated white colony from each plate and propa-
gate the BACs for sequencing and virus reconstitution.
3.3 Construction
of Recombinant
Adenoviruses by Flp
Recombination in
E. coli
1. In this protocol the gene of interest is cloned into a shuttle
plasmid, which is unifi ed with a modifi ed Ad-BAC by Flp
recombination in
E. coli
via their FRT sites (
see
Figs.
2a, b
).
Therefore, the gene of interest, or the ORF to be expressed,
needs to be cloned fi rst into the vector pO6A5-CMV (
see
Note
15
and Fig.
2c
).
2. Retransform the FRT ready Ad-BAC vector (pBA5-FRT,
see
Note 16
) to DH10B strain of
E. coli
(
see
Note 17
) or plate out
a small aliquot of frozen stock of the FRT-containing Ad-BAC
vector on LB plates containing 25
g/mL chloramphenicol.
3. Prepare electrocompetent cells from a single colony of DH10B
cultures of the pBA5-FRT (
see
Note 18
).
4. Transform the competent DH10B cells carrying the pBA5-
FRT with 5 ng of pCP20 [
13
] (
see
Note 19
) and plate out
10
μ
μ
L the 1 mL outgrowth cultures on LB plates containing
25
g/mL ampicillin and incubate
them overnight at 32 °C. Alternatively, plate out a small ali-
quot of frozen stock of the DH10B cells carrying both the
pBA5-FRT and pCP20 on LB plate containing 25
μ
L chloramphenicol and 50
μ
μ
g/mL
g/mL ampicillin and incubate them
at 32 °C overnight (
see
Note 20
).
5. Inoculate 5 mL LB containing 25
chloramphenicol and 50
μ
μ
g/mL chloramphenicol
and 50
μ
g/mL ampicillin with a single colony from the plates