Biology Reference
In-Depth Information
Serum-Free DMEM.
0.05 % Trypsin-EDTA solution (1×) (Life technologies, cat. no.
25300-054).
Sterile phosphate-buffered saline (PBS) without calcium chloride
and magnesium chloride (Sigma, cat. no. D-8537).
Plasmids: pDG [helper plasmid containing AAV2 rep and cap
genes and the adenovirus helper genes (E2A, E4orf6, VA
RNA)] [ 48 ], rAAV (expression plasmid containing the gene of
interest flanked by AAV2 ITRs).
Polyethylenimine (PEI), linear, MW 25,000 (Polysciences, cat. no.
23966).
Lysis buffer: 150 mM NaCl, 50 mM Tris-HCl, pH 8.5.
Cell culture disposable plastic ware: T-225 cell culture flask (BD
Biosciences), cell culture triple flask (Thermo Scientific, cat.
no. 132913), and 250 ml conical tubes (Corning, cat. no.
430776).
Dry ice/ethanol mix.
4.9 M MgCl 2 (Sigma, cat. no. 104.20).
Benzonase (250 U/μl, Sigma, cat. no. E1014).
Iodixanol, Optiprep 60 % (w/v) (Sigma, cat. no. D1556).
Phenol red solution 0.5 % (Sigma, cat. no. P0290).
Vivaspin 20 concentrator, 100 kDa (Sartorius Stedimcat, cat. no.
VS2041).
Lactated Ringer's Solution (Fisher Scientific, cat. no. W4915E).
Conical tubes (15 ml, 50 ml, and 250 ml).
Sterile alcohol wipe (Kendall Webcol).
3-ml syringes (Becton Dickinson, cat. no. 300910) and 18G × 1½ in.
needles (Becton Dickinson, cat. no. 301500).
37 °C water bath.
Stand and clamp (Fisher Scientific).
Ultracentrifuge equipped with 70Ti rotor (Optima L90K, Beckman
Coulter).
Optiseal polyallomer Beckman tubes (Beckman Coulter, cat. no.
361625).
Ultem tube spacer (Beckman Coulter, cat. no. 361669).
2.3 Purification
of AAV
2.4
Vector Titration
(a) Quantitative PCR and Dot Blot assay
(α) DNase I treatment
DNase I (Sigma, cat. no. AMPD1)
DNase I buffer: 10 mM Tris-HCl, pH 7.5; 1 mM MgCl 2 .
Search WWH ::




Custom Search