Biology Reference
In-Depth Information
2
Materials
2.1 Adenovirus
Construction
by Homologous
Recombination
in Bacteria
1. LB Broth: 25 g Miller's LB in 1 L of ddH
2
O. Sterilize by
autoclave.
2. LB + Ampicillin: Add 100 mg ampicillin to 1 L LB Broth.
3. LB + Ampicillin plates: Add 15 g agar to 1 L LB Broth.
Autoclave. Cool down to 50 °C and add 100 mg ampicillin.
Pour on plates.
4.
E. coli
strain BJ5183 (endA,
sbcB
−
, recBC
−
, str
R
).
5.
E. coli
strains DH5
or similar.
6. Commercial DNA kit for purifying DNA fragments from aga-
rose gels.
7. 1 % (wt/vol) agarose gels.
8. Appropriate restriction enzymes.
9. Commercial DNA minipreparation kit.
10. Spectrophotometer.
α
2.2 Generation
of Recombinant
Adenovirus
1. Dulbecco's Modifi ed Eagle's Medium (DMEM) supplemented
with 10 or 2 % fetal bovine serum (FBS).
2.
Pac
I restriction enzyme.
3. TE buffer (10 mM Tris-HCl, 1 mM EDTA).
4. HEK-293 cells or other adenovirus packaging cell lines.
5. 10 mM polyethylenimine (PEI) 25,000, branched (Sigma, ref.
408727).
6. 150 mM NaCl.
2.3 Purifi cation
of Recombinant
Adenovirus by
Banding on CsCl
1. Ultracentrifuge: Beckman Coulter Optima L90 K or L100XP
and SW32Ti and SW40Ti rotors (Beckman Coulter). Ultra-
Clear centrifuge tubes for SW32Ti rotor (Beckman, ref.
344058) and polyallomer centrifuge tubes for SW40Ti rotor
(Beckman Coulter ref. 331374).
2. CsCl solutions of densities: 1.4 g/mL, 1.34 g/mL, and
1.25 g/mL in TD buffer (137 mM NaCl, 5.1 mM KCl,
0.7 mM Na
2
HPO
4
·7H
2
O, 25 mM Tris Base, pH = 7.4 (adjusted
with HCl).
3. 18 G needles, 10 mL syringes, pipette-aid, 10 mL pipettes.
4. Disposable Sephadex G-25 columns (PD-10, GE Healthcare,
ref. 17-0851-01 or equivalent).
5. 1× PBS Ca
++
/Mg
++
(PAA ref. H15-001).
6. Glycerol anhydride.
