Biology Reference
In-Depth Information
Gel fixing solution: 50 % ethanol, 15 % (v/v) acetic acid in
ultrapure water.
Destaining solution: 5 % (v/v) acetic acid, 0.1 % (v/v) Tween ® -
20 in ultrapure water.
Protein electrophoresis (Mini-Protean, Bio-Rad, cat. no.
165-8000).
LI-COR/Odyssey infrared imaging system
(b) Endotoxin assay
Limulus Amebocyte Lysate (LAL) kit (Lonza, cat. no. 50-647U).
LAL reagent water (Lonza, cat. no. W50-100).
Stop reagent: acetic acid, 25 % (v/v) glacial acetic acid in ultra-
pure water.
Disposable endotoxin-free glass dilution tubes (Lonza, cat.
no. N207)
96-well microplate.
Dry block heater at 37 °C.
Microplate absorbance reader (405-410 nm filter) (Bio-Rad,
iMark, cat. no. 168-1135)
(c) Replication-competent AAV detection
HeLa or 293T cells.
cf infectious center assay for materials and reagents.
3
Methods
3.1 Plasmids
Characterization
The AAV ITRs are prone to rearrangements in plasmids propa-
gated in commonly used E. coli strains (e.g., DH5α). This instabil-
ity can lead to partial or even complete deletion of the ITR resulting
in low rAAV vector yield. To overcome this issue, rAAV vector
plasmids are propagated at 30 °C in recombination-deficient E. coli
strains such as the SURE. The integrity of the AAV ITR is checked
for each rAAV vector plasmid preparation by restriction digest with
an enzyme that cut inside the ITRs (e.g., SmaI).
The DNA used for AAV vector production is purified using
endotoxin-free plasmid kits, according to the manufacturer's
instructions. DNA concentration is determined using an UV spec-
trophotometer, and only DNA preparations with a 260/280 ratio
greater than 1.8 are used for transfections.
3.2 rAAV Virus
Production
(a) Seeding a triple flask
The day before transfection, seed low-passage HEK_293T
cells into a triple flask from a confluent T-225 flask.
Aspirate media and carefully wash cells twice with 15 ml of PBS.
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