Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 30

Proteinase K solution (Sigma, cat. no. P4850)
2× Proteinase K buffer: 20 mM Tris-HCl, pH 8.0; 20 mM
EDTA, pH 8.0; 1 % SDS.
Phenol/chloroform/isoamyl alcohol (25:24:1) (Sigma,
cat. no. P2069)
3 M sodium acetate (Sigma, cat. no. S7899).
Glycogen (Roche Applied Science, cat. no. 901393).
100 % and 70 % Ethanol.
TE: 10 mM Tris-HCl, 1 mM EDTA.
(β) Quantitative PCR
SYBR Green JumpStart Taq Ready Mix without MgCl 2
(Sigma, cat. no. S5193).
Water, PCR reagent (Sigma, cat. no. W1754).
Reverse and forward primers at 20 μM final each.
MgCl 2 25 mM, PCR reagent (Sigma, cat. no. M8787).
2-4 % agarose gel, 1× TAE, electrophoresis apparatus.
Lightcycler capillaries (Roche Applied Science, cat. no.
04929292001).
Lightcycler 2.0 instrument (Roche Applied Science, cat.
no. 03531414001).
(γ) Dot blot assay
Denaturing solution: 0.4 M NaOH, 10 mM EDTA.
2× SSC: 0.3 M NaCl, 30 mM sodium citrate, pH 7.0.
a- 32 PdCTP, EasyTides ® Deoxycytidine, 6,000 Ci/mmol
(Perkin Elmer, cat. no. NEG513Z250UC).
Prime-It RmT Random Primer Labeling Kit (Stratagene,
cat. no. 300392).
G50 spin columns (Roche Applied Science, cat. no.
11273973001).
0.75×, 0.5×, 0.1× NW buffer.
Nylon membrane, Hybond-XL (Amersham, cat. no.
RPN303S).
Filter papers (Bio-Rad, cat. no. 1620161).
3MM chr blotting paper (Whatman).
Slot blot apparatus (Bio-Rad, Bio-Dot apparatus, cat. no.
170-6542).
Heat block at 100 °C.
UV crosslinker CL-1000 utilizing a 254-nm light bulb
(Ultra Violet Products, cat. no. 95-0174-02).
Scintillation counter (Beckman Coulter LS 6500).
Hybridization incubator (Ultra Violet Products, cat. no.
95-0030-02).