Gene Therapy for Huntington’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 143

87.5 μL PCR grade water per reaction. Incubate for 15 min at
25 °C to remove any contaminating DNA not packaged within
rAAV virions. All incubation steps were carried out in a ther-
mocycler with a 0.5 mL block.
2. Add 1 μL of 100 mM EDTA. Vortex the contents of tubes and
spin briefly before heat inactivating the DNase I at 70 °C for
10 min.
3. Digest the viral capsids by addition of 99 μL of proteinase K
master mix consisting of 1 μL proteinase (14-22 μg), 10 μL
10× DNase I buffer, and 88 μL PCR grade water per reaction.
Vortex samples to mix and centrifuge briefly before incubation
at 55 °C for 1 h.
4. Inactivate the proteinase K by heating at 95 °C for 20 min.
Store samples at 4 °C until plate preparation. For consistency
of results, vector DNA extraction and real-time PCR should be
carried out on the same day. However, should the need arise,
vector DNA samples or plates prepared for real-time PCR can
be stored short term at 4 °C.

1. Use a plasmid of known size containing the amplifiable sequence
to generate a dilution series of 10 2 -10 7 copies/μL. Dilute the
plasmid to 25-100 ng/μL using sterile 1× TE or PCR grade
water and determine the exact concentration using a high-
accuracy spectrophotometer. Use the following equation to
determine the mass of a single plasmid molecule ( M ):

3.3.2 Quantitative
Real-Time PCR

æ ö g

1mole æ

ç

660
mole

ö
÷

MN =

ç ÷

()

6 023 10 23

. ´ moleculesbp

è ø

æ
ç

g
bp

ö
÷

1 096 10 21

-

MN = . ´

where N = DNA size (bp)
Avogadro's number = 6.023 × 10 23 molecules/1 mol
Average MW of a double-stranded DNA molecule =
660 g/ mol
Using this number, determine the concentration in copies/
μL of your plasmid stock. Dilute this to generate a 10 9
copies/μL stock. Make a tenfold serial dilution of this stock to
generate the titering standards and store as small aliquots at
−20 to −80 °C. Thaw a new set of standards for each PCR run.
2. Vortex and briefly spin vector DNA samples (Sect. 3.3.1 ).
Dilute a 4 μL sample of each triplicate vector DNA preparation,
50-fold, by the addition of 196 μL PCR grade water. Vortex
and briefly spin samples. Depending on the concentration of
the vector preparation, the level of dilution should be adjusted
such that the quantity of rAAV vector genome amplicons
generated falls within the linear range of the standard curve.