Gene Therapy for Epilepsies - Gene Delivery and Therapy for Neurological Disorders - page 248

Fig. 1 Flowchart outlining the main steps in the AAV production

2. Three hours before transfection, remove the media and replace
with 18 ml pre-warmed (in 37 °C water bath or incubator)
IMDM/5 % FBS. Take out 2× HeBS buffer, 2.5 M CaCl 2 , and
distilled water to warm up to room temperature.
3. Make up the transfection mix using tissue culture grade water
in a 50-ml disposable polypropylene conical tube in the follow-
ing order: water, CaCl 2 , plasmid. Mix well after addition of each
component. When transfecting multiple dishes, multiply vol-
umes given below by the number of dishes to be transfected.

Plasmids and reagents Amount per 15-cm dish

pFdelta6 27 μg

pNLrepcap1 9 μg

AAV pAM packaging
plasmid

9 μg

2.5 M CaCl 2 100 μl

Distilled water Make up mix to

1,000 μl