Biology Reference
In-Depth Information
12. Centrifuge in a Beckman SW41Ti rotor at 32,000 rpm
(176,000 ×
g
) for 20 h at 4 °C.
13. After centrifugation usually only one band comprised of the
HC-Ad vector is visible. This band should be collected by
punctating the tube as described in
step 9
of the purifi cation
protocol. If more than one band is visible each band should be
collected separately by punctating the tube at the respective
position starting with the band with the lowest density (
see
Note 17
). Take care to collect the individual bands in not
more than 2 mL. If more than one band is present in the gradi-
ent and collected the following steps of the protocol have to be
performed separately for each band.
14. Transfer the collected band to a new reaction tube and fi ll up
with HEPES 50 mM pH 8.0 to a fi nal volume of 2.5 mL. Place
the tube on ice.
15. Equilibrate a disposable PD-10 desalting column with 5 × 5 mL
HEPES 50 mM pH 8.0 (
see
Note 18
).
16. Load the HC-Ad vector solution (2.5 mL) onto the PD-10
column. Discard the fl ow-through.
17. Elute the HC-Ad vector from the PD-10 column with 5 mL
HEPES 50 mM pH 8.0. Collect the eluate in 1 mL fractions.
18. Combine fractions 2 and 3 of the eluate, which contain the
HC-Ad vector and add 222
L 100 % glycerol. Mix carefully
by pipetting up and down. Remove a 100
μ
L aliquot from the
HC-Ad vector preparation for analysis of the vector DNA and
a 20
μ
L aliquot for a preliminary titer determination by OD
measurement.
19. Aliquot the HC-Ad vector and store at −80 °C.
μ
3.5 Characterization
of the HC-Ad Vector
Genomes
To prove the integrity of the HC-Ad vector genomes it is mandatory
to isolate the vector DNA from the particles and perform a
restriction digest with at least two different enzymes. As a control
100-200 ng of the linearized vector plasmid from
step 3
of the
rescue protocol is digested with the same enzyme in parallel.
1. Isolate vector DNA from the 100
L aliquot obtained in
step
18
of the purifi cation protocol using the QIAamp DNA Mini
Kit according to the manufacturer's instructions.
2. Digest an aliquot of the isolated vector DNA with an appropri-
ate restriction enzyme. As a control digest 100-200 ng of the
linearized vector plasmid from
step 3
of the rescue protocol
with the same enzyme.
3. Load the samples on an agarose gel, perform ethidium bro-
mide staining and identify the vector bands (
see
Note 19
).
μ
