Gene Therapy for Huntington’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 137

Table 3
Solutions and reagents

Solutions

Heparin column purification buffers

5 M NaCl ( mL ) 1 M Tris, pH 8

( mL )

Final volume ( mL )

100 mM NaCl, 20 mM Tris, pH 8 10 10 500

200 mM NaCl, 20 mM Tris, pH 8 2 1 50

300 mM NaCl, 20 mM Tris, pH 8 3 1 50

400 mM NaCl, 20 mM Tris, pH 8 4 1 50

450 mM NaCl, 20 mM Tris, pH 8 4.5 1 50

500 mM NaCl, 20 mM Tris, pH 8 5 1 50

Add required amount of 5 M NaCl and 1 M Tris, make up to a final volume with ultrapure water, and
adjust pH to 8 with HCl. Filter sterilize and store at 4 °C

10 × PBS - MK

Dissolve 0.2 g MgCl 2 · 6H 2 O in 100 mL 10× PBS. Filter sterilize and store at room temperature. Dilute
10× stock to 1× PBS-MK with ultrapure water where required, filter sterilize, and store at 4 °C

1 % pluronic acid

Dissolve 0.5 g Pluronic F68 (Fluka, #81112) in 50 mL ultrapure water. Filter sterilize and store at
4 °C. Add to 1× PBS-MK to a final concentration of 0.001 %

Reagent Supplier Item number

HiTrap ® Heparin Cartridge Sigma 54836

Amicon Ultra 4 100K MWCO centrifugal
filter units

Millipore UFC810024

Acrodisc syringe filters 13 mm 0.2 μm
Tuffryn

Pall 4454

For chimeric AAV1:2 vectors

12 mL ultrapure water
1.65 mL 2.5 M CaCl 2
62.5 μg pAAV expression plasmid
125 μg pFdelta6 (adenovirus helper plasmid)
30 μg pNLrep (AAV2 helper)
31.25 μg pH21 (AAV1 capsid)

For AAV vectors (any serotype)

12 mL ultrapure water
1.65 mL 2.5 M CaCl 2
62.5 μg pAAV expression plasmid (AAV cis plasmid)
125 μg pFdelta6 (adenovirus helper plasmid)