Biology Reference
In-Depth Information
UltraClear tube (14 × 89 mm, 12.8 mL total volume). If more
cells are used (for example, in a large-scale preparation) accord-
ingly more UltraClear tubes are required.
15. CsCl solutions should be prepared in an appropriate buffer sys-
tem and sterile fi ltered. We have excellent experience using
50 mM HEPES as buffer system. In that case, the storage
bottles should be wrapped into aluminum to protect from
light. Alternative buffer systems, e.g., 100 mM Tris may be
used. The solutions can be stored for prolonged time at room
temperature, but each solution should be mixed prior to use.
It is recommended to verify the density of each solution by
weighing 1 mL immediately prior to use. To prepare 100 mL
of a CsCl solution with a density of 1.27 g/mL dissolve
36.94 g CsCl in a fi nal volume of 100 mL 50 mM HEPES
pH 8.0. To prepare 100 mL of a CsCl solution with a density
of 1.34 g/mL dissolve 47.2 g CsCl in a fi nal volume of 100 mL
50 mM HEPES pH 8.0. To prepare 100 mL of a CsCl solu-
tion with a density of 1.42 g/mL dissolve 56.78 g in a fi nal
volume of 100 mL 50 mM HEPES pH 8.0.
16. Clearing the cell lysates prior to loading onto the CsCl step
gradients is mandatory. If the cell lysates are not cleared by
centrifugation before loading onto the gradient, the gradient
will be disturbed and the purity of the vector preparation will
be low. It is recommended to keep the cell debris (supple-
mented with 10 % glycerol and stored at −80 °C), which still
contains signifi cant amounts of HC-Ad vector since it can be
used for reinfecting fresh producer cells for another round of
vector production.
17. The appearance of more than one band in the continuous CsCl
gradient can have several causes. First, one additional band can
be comprised of helper virus. Second, additional bands can be
comprised of rearranged vector genomes. In any case the bands
should be collected separately, DNA should be prepared from
the vector particles and analyzed by restriction digestion and
agarose gel electrophoresis and/or Southern blotting. It is not
recommended to use a HC-Ad vector preparation for which
several bands occurred in the continuous gradient for the rein-
fection of fresh producer cells.
18. Alternative protocols use dialysis as the preferred method for
desalting HC-Ad vector preparations. Compared to dialysis
the disposable PD-10 desalting columns are time saving.
19. DNA isolated from Ad vector particles can be diffi cult to
digest. It is recommended to use robust enzymes and perform
overnight digestions. The linearized plasmid control will
