Biology Reference
In-Depth Information
6. The heat shock at 42 °C will induce expression of the lambda
phage derived red recombinases. It is crucial to keep this induc-
tion time as rigorously as possible for reproducible results.
7. Make sure that MacConkey plates are prepared using premixed
agar powders, which do not contain any sugar and thus, added
galactose will be the only carbon source in the plate. We rec-
ommend using MacConkey agar from Difco.
8. It is crucial to produce pure stocks of recombineering interme-
diate construct for the second targeting. Do not save time
avoiding this replating step(s). A few germs carrying the origi-
nal target BAC contaminating the stocks of the intermediates
can corrupt the second targeting!
9. As BACs are propagated in
E. coli
in single copy, typical yields
of BAC DNA are usually only between 10 and 30
g from a
100 mL overnight culture. We recommend using Nucleobond
PC-20 and PC-100 (Macherey and Nagel) for small and mid-
dle scale preparations, respectively. The small-scale Nucleobond
PC-20 column yields preparations suffi cient for 2-3 restriction
analysis and retransformation. Sequencing, detailed restriction
analysis, or virus reconstitution requires larger scale Nucleobond
PC-100 purifi cation. The culture volume here can be scaled up
to 200 mL if multiple assays are planned.
10. It is very valuable to have a pure, well-characterized recom-
bineering intermediate construct. The work above can be
saved for the next mutation within the same region if the inter-
mediate construct is kept as a glycerol stock.
11. To clone the genomic adenovirus DNA as BAC, viral DNA can
be purifi ed either from the infected cells or purifi ed virions [
1
,
3
]. For this recombineering, a special targeting BAC construct
is required, which needs to be constructed by traditional clon-
ing procedures. This intermediate construct should contain
the galK-Kn cassette fl anked by the left and the right ends (few
100 nt each) of the Ad genome of interest [
1
,
3
]. See the tar-
geting construct pB-TA5 for cloning wild-type Ad5 genomes
and/or its variants in Fig.
1d
as an example. The pB-TA5 is
available upon request from the corresponding author.
12. One can inoculate overnight culture from a glycerol stock
using fi tting intermediates from the previous experiment. In
this case the recombination fragment needs to be prepared to
fi t to the previous intermediate.
13. As a rule of thumb, more DNA is needed if larger fragments
are introduced. To start with 500 ng insert for a standard
recombineering normally delivers good results. However,
DNA amounts may be limited by the toxicity after transforma-
tion or by arcing during the electroshock. In these cases the
DNA amounts need to be systematically reduced.
μ
