Adeno-Associated Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 15

6. After centrifugation the tubes are clamped in a retort stand
and a 19 gauge needle is inserted into the top of the tube. The
clear fraction of the 40 % layer now contains the virus (Fig. 1b ).
In order to extract the pure virus sample and to separate it
from the debris and empty capsids, the best method to employ
is fractionation. For this, a standard 19 gauge (or slightly thin-
ner) syringe needle is inserted approximately 1 cm from the
bottom of the tube. It should be pushed half way into the tube
with the bevel pointing upwards. The placing of the needle
should be such that it is towards the upper portion of the 60 %
iodixanol layer. It is important to remember to puncture the
top of the tube first so as to allow the liquid to flow. Placing
the needle in this manner should cause slow leakage of the
solution out of the tube in a drop-wise fashion. The drops are
then collected in samples of approximately 250 ʼl in 1,500 ʼl
Eppendorf tubes. The entire 40 % layer and the first portion of
the 25 % layer are collected.
7. This process is repeated for subsequent tubes and the collected
fractions may be stored at 4 °C until further analysis provided
it is carried out within a few days.
8. 4 ʼl of each fraction is diluted with water, combined with 4×
reducing loading buffer, boiled and run on 10 % SDS-
PAGE. Capsid proteins of AAV are visualized by SYPRO Ruby
staining and fractions containing pure virus are pooled (Fig. 2 ).
Only the fractions containing bands for the three capsid pro-
teins are pooled as high-quality virus. Fractions with some
higher molecular weight bands may also be pooled separately
to be used in in vitro validation studies.

1. AAV is concentrated and desalted by centrifuging through a
BIOMAX 100 Ultrafree 15 centrifugal filter device (Millipore
UFV2BHK 10 or 40) or Amicon Ultra centrifugal filter device
(Millipore UFC910008).

3.9 Concentration
and Desalting of AAV
Preparations

Fig. 2 Viral sample fractionation. Collected fractions were analyzed on 10 %
polyacrylamide gel stained with SYPRO Ruby; fractions collected here were all
pooled as high-quality virus as no contaminating bands were detected. These
collected fractions were then taken for further concentration