Biology Reference
In-Depth Information
in suspension culture for 72-96 h amplification cycles. In order to make P2 virus, a
suspension culture in 24-well deep blocks or six-well dishes of 4 ml of
0.5-1
10 6 cells/ml were infected with 200-400 m l of P1 virus and incubated for
72-96 h (at an MOI of 0.1-0.2). P2 was harvested by centrifugation and 500 m lto
1 ml was used to infect 50 ml of cells at 3
10 6 cells/ml in order to generate P3 virus
(at an MOI of 15-30). Supernatants from P3 suspension cultures were used to infect
either Sf9 or Hi5 cells at a density of 1
10 6 cells/ml at a multiplicity of infection of
1-10 for large-scale protein production. Yield for FITM2 protein was 2-8 mg/l of
culture using either system, with higher yields usually obtained for Novagen's
pIEx/Bac-1-BacMagic 2 system compared to Invitrogen's pFastBac1-Bacmid
system.
11.2.1.4 Bacmid purification and storage
In order to purify large quantities of recombinant bacmid from PCR-confirmed colo-
nies, we inoculated corresponding DH10Bac E. coli colonies into 6 ml of Luria-
Bertoni (LB) broth with appropriate antibiotics (no BluoGal), and incubated overnight
at 37 C while shaking at 250 rpm. After 18-20 h incubation, 1 ml was removed to
generate a glycerol stock (30-40% glycerol (w/v)) and stored at
80 C. We then
harvested the remaining 5 ml of cells at 16,000
g for 5 min and discarded the
supernatant. The pellet was resuspended in 400 m l of resuspension buffer (50 mM
Tris-HCl, pH 8.0, 10 mM EDTA, 100 m g/ml RNase A) and vortexed until cells were
resuspended. Subsequently, 400 m l of lysis buffer (200 mM NaOH and 1% SDS) was
added, the tube was inverted 10 times, and then incubated at room temperature for
5 min. After incubation, 400 m l of neutralization (N3) buffer (2.8 M potassium acetate
pH 5.1) was added and the tube was again inverted 10 times, but carefully this time
around. All subsequent handling steps were performed with utmost attention not to
shear the bacmid DNA which was no longer intracellular. The precipitate was centri-
fuged at 16,000
g for 20-30 min at 4 C and transferred to a fresh Eppendorf tube.
Supernatant was centrifuged again for 10-15 min at 16,000
g at 4 C. The second
supernatant was split into two tubes and 700 m l of DNA-grade isopropanol was added
to each with gentle agitation in order to precipitate the bacmid DNA on ice for 30 min.
The isopropanol-precipitated DNA was isolated by centrifugation at 16,000
g for
15 min at 4 C. The supernatant was carefully pipetted off without disturbing the pellet
and 1 ml of 70% ethanol in deionized water was added to the tube, which was gently
inverted several times and centrifuged again at 16,000
g for 15 min at 4 C. The
ethanol-containing supernatant was pipetted off carefully and the pellet dried for no
more than 5 min. Care must be taken to avoid overdrying the pellet, as this makes
it very difficult to resuspend in aqueous solution for subsequent manipulations. The
pellet was resuspended in 100 m l of TE buffer (Tris-EDTA, pH 8) without pipetting
as pipetting will shear the bacmid DNA which is
100 kb. A spectrophotometer was
then used in order to determine concentration of the bacmid, and 0.5-0.7% agarose gel
electrophoresis was utilized to determine bacmid purity. Purified bacmid was stored at
4 C for 1-2 weeks without significantly affecting protein yield. If more bacmid needs
>
Search WWH ::




Custom Search