Biomedical Engineering Reference
In-Depth Information
4 Discard the supernatant and resuspend cell pellet in 2ml PBS
++
plus 10% glycerol per
T175 flask harvested.
5 Freeze at
−
70
◦
Cuntillysisisrequired.
6 Thaw the cellular material and lyse with a 5% solution of sodium deoxycholate (add
1.5 ml solution/15ml lysate).
7 Add 75
μ
lof1mg/mlDNaseIand300
μ
l1
M
MgCl
2
for every 15ml lysate, and incubate
at 37
◦
Cfor1h.
d
8 Centrifuge at 1000
g
15min.
9 Discard the pellet and filter sterilize the supernatant through 0.45
μ
mlow-protein
syringe filters.
10 Confirm there is adenovirus in the crude lysate by FG293 cell plaque assay (see Protocol
11.2) and a suitable functional assay to check that the protein of interest is
expressed.
11 Aliquot and store at
−
70
◦
C.
Notes
d
You will need 75
μ
l DNase I solution for every 15ml lysate.
PROTOCOL 11.4 Preparation of CsCl
2
Purified
Recombinant Adenovirus
Equipment and reagents
•
Class II safety cabinet
•
Ultracentrifuge and fixed-angle rotor
•
Ultracentrifuge tubes and sealing assembly
•
70% ethanol
•
Sterile distilled H
2
O
•
Glass Pasteur pipettes
PBS++ (PBS supplemented with 0.01% CaCl
2
and 0.01% MgCl
2
)
•
CsCl
2
solution,
ρ
= 1.34: 51.2 g CsCl
2
dissolved in 100ml buffer DG (750m
M
NaCl; 50 m
M
KCl; 250m
M
Tris-HCl, pH 7.4)
•
CsCl
2
solution,
ρ
= 1.40: 62 g CsCl
2
in 100ml DG
•
Sterile 21-gage needles
•
Sterile 2ml syringes
•
Slide-a-lyse dialysis cassette (Pierce; Perbio Science UK Ltd)
•
