Optiprep
5 M NaCl
5×TD
Water
Final volume
15 %
45 ml
36 ml
36 ml
63 ml
180 ml
30 %
60 ml
–
24 ml
36 ml
120 ml
40 %
68 ml
–
20 ml
12 ml
100 ml
60 %
100 ml
–
–
–
100 ml
4.
Benzonase nuclease (Novagen, San Diego, CA, USA; or Sigma-Aldrich, St Louis, MO, USA).
5.
Ultracentrifuge and rotor.
6.
Ultracentrifuge tubes: Sorvall 35 ml, ultracrimp tubes for rotor T-865 (ThermoFisher Scientific, Waltham, MA, USA) or Beckman Optiseal tubes for rotor Ti70 (Beckman Coulter, Pasadena, CA, USA).
7.
Apollo 20 ml centrifugal quantitative concentrators with 150 kDa Molecular cutoff (Orbital sciences, Topsfield, MA, USA).
2.4 Viral Vector Preparation for Titering
1.
DNase I 2 U/μl (Ambion/Life technologies, Grand Island, NY, USA).
2.
10× DNase buffer: 100 mM Tris pH 7.5, 25 mM MgCl2, 5 mM CaCl2.
3.
Proteinase K (Ambion/Life technologies, Grand Island, NY, USA).
4.
Proteinase K buffer: 100 mM Tris pH 8100 mM EDTA,10 % SDS.
5.
Lactated Ringers solution or PBS.
6.
Glycoblue (Life Technologies, GrandIsland, NY, USA).
2.5 Viral Vector Quantification (Two Different Methods: Real-Time PCR and Dot Blot): Real-Time PCR
1.
RT PCR primers: Designed to specific construct or promoter following standard molecular procedures.
2.
DNA standards (preferably from the plasmid used to make your virus ) of 500, 100, 20, 5, 1, and 0.2 pg (see Note 6 ).
3.
Viral DNA samples. Run your viral samples in triplicate.
4.
Sybr green master mix (2×) (Takara, Mountain View, CA, USA).
2.6 Viral Vector Quantification (Two Different Methods: Real-Time PCR and Dot Blot): Dot Blot
1.
1× Alkaline buffer: 0.4 M NaOH, 10 mM EDTA.
2.
Hybridization buffer: 7 % SDS, 1 mM EDTA, 0.25 M NaHPO4 pH 7.2.
3.
Wash buffer: 1 % SDS, 1 mM EDTA, 40 mM NaHPO4 pH 7.2.
4.
DNA probe: 1 mM each dGTP, dCTP, dTTP, plus 0.84 mM dATP and 0.16 mM biotinylated dATP (Enzo Life Sciences, Farmingdale, NY, USA) (see Note 7 ).
5.
Hybridization oven and hybridization tubes.
6.
Hybond-N membrane (GE Life Sciences, Pittsburgh, PA, USA).
7.
Whatman filter paper.
8.
UV cross-linker.
9.
Streptavidin-conjugated antibody: Streptavidin IRDye-800CW (LI-COR Biotechnology, Lincoln, NE, USA), or equivalent.
10.
LI-COR Odyssey scanner (LI-COR Biotechnology, Lincoln, NE, USA), or equivalent.
3 Methods
Carry out all procedures under sterile conditions in a biosafety cabinet.
3.1 Tissue Culture
1.
Human embryonic kidney-derived 293B cells (HEK 293) will be grown on 15 cm tissue culture dishes. Cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 ° C in a 5 % CO2 incubator. First thaw cells and add to a 10 cm tissue culture dish. Grow until they are confluent. Then pass them to a 15 cm dish and let them grow until confluent. The volume of added medium for a 15 cm plate is 20 ml.
2.
10 plate prep: Grow cells to confluence in a 15 cm dish and then passage the cells 1/10 to generate 10 × 15 cm plates from each original 15 cm plate. If cells are healthy the plates should be ~70 % confluent and ready for transfection after 3 days.
3.
20 plate prep: Keep passing 15 cm dish until you have 12 dishes. Pass cells in the 12 dishes 1:2 once they are confluent, so they will be ready for transfection the following day (see Note 8 ).
3.2 Calcium Phosphate Transfection (Method 1)
1.
Check plates. Cells should be 70 % confluent the day of transfection .
2.
Thaw 2× HBS and keep at 37 °C until ready to use.
3.
Make CaCl2 solution.
4.
Calculate the amount of DNA needed for transfection : Helper DNA containing adenoviral and rep and cap AAV functions are transfected at a 1:1 molar ratio. For 20 dishes you need 900 μg of helper (Ad and rep and cap helper function plasmid ) and 300 μg of rAAV. Add water to a 50 ml conical tube followed by 2.5 ml CaCl2 (final CaCl2 concentration = 0.25 M), and then add plasmid DNAs. The total volume should be 25 ml (see Note 9 ). If performing a triple transfection, e.g., using pXX6 (Ad helper DNA) with a pAAV (rep and cap helper DNA), the following amounts of DNA are used. For 20 dishes rAAV vector (300 μg), pAAV serotype plasmid (300 μg), and pXX6 Ad helper plasmid (700 μg) (see Note 10 ).
5.
Add CaCl2 and DNA mixture to the conical tube containing 25 ml of 2× HBS dropwise and bubble air through the 2×HEPES solution with a 2 ml serological pipette hooked to a pipet aid while adding the DNA/CaCl2 solution dropwise with a P1000 pipetman. Do not vortex transfection cocktail.
6.
Add transfection mix to cells with media dropwise, 2.5 ml per 15 cm plate.
7.
Incubate at 37 °C for 48–65 h.
3.3 PEI Transfection (Method 2)
1.
Prior to transfection heat PI 55 °C to dissolve clear.
2.
The amounts of DNA outlined above in step 4 in Subheading 3.2 (calcium phosphate transfection ) can be used here and added to a sterile 50 ml tube.
3.
To the DNA also add 2.5 ml of 1.5 M NaCl and sterile water to a final volume of 25 ml.
4.
In a second tube mix by vortexing briefly the following: 8 ml PEI (0.323 g/L), 2.5 ml 1.5 M NaCl, and 14.5 ml water.
5.
Add PEI mix to DNA mix and briefly vortex for ~20 s.
6.
Incubate at room temperature for 15 min.
7.
Add DNA /PEI mixture to cells: 2.5 ml/15 cm dish.
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