Biology Reference
In-Depth Information
10. TE buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.
11. Elution buffer: 100 mM NaHCO
3
, 1 % SDS.
12. Salmon sperm DNA-protein A Agarose (Millipore Cat. #
16-157).
13. Polyclonal or monoclonal antibodies.
14. 20 mg/mL Protease K (NEB Cat. # P8102S).
15. 5 M NaCl.
16. 0.5 M EDTA.
17. 1 M Tris-HCl, pH 6.5.
18. 10 mg/mL Glycogen.
19. Phenol/Chlorofrom.
20. 3 M Sodium Acetate (NaOAc), pH 5.2.
21. DyNAmo™ HS SYBR
®
Green qPCR Kit (Thermo Cat. #
F-410 L).
22. DNase/RNase-Free Distilled Water.
1. Branson Sonifi er 450 with 1/8” microtip.
2. DNase/RNase-free barrier tips.
3. Applied Biosystems 7500 Real-Time PCR system (or equiva-
lent quantitative PCR machine).
4. MicroAmp
®
Optical 96-Well Reaction Plate (Invitrogen Cat.
# 4316813).
5. MicroAmp
®
Optical Adhesive Film (Invitrogen Cat. #
4311971).
2.3 Equipment
and Other Materials
3
Methods
3.1 Cultured Cells
and Virus Infections
Any cell line that is permissive for Ad infection may be used in
these analyses. In our work, we utilized N52.E6 cells which
express Ad E1A and E1B proteins [
15
]. Cells were grown in
alpha modifi cation of Eagle medium supplemented with 10 %
bovine fetal serum, 2 mM glutamine, penicillin, and streptomy-
cin. Approximately 10
7
cells are found per 100 mm dish. Include
one extra dish to be used solely for estimation of cell number.
Purifi ed Adenovirus particles were prepared by CsCl equilibrium
gradient centrifugation.
1. Cells were infected with 100 virus particles/cell.
2. After 1 h absorbation at 37 °C, remove the virus inoculum,
wash the cells twice, and add fresh medium.
3. Incubate infected cells at 37 °C for 18-24 h, although any
suitable time point may be used depending on the nature of
the analysis.
