Biology Reference
In-Depth Information
2. Incubate the blank and the adenovirus samples for 10 min at
56 °C.
3. Centrifuge 1 min at 16,000 ×
g
.
4. Measure the optical density at 260 nm.
5. The concentration of adenoviral particles is determined by
multiplying the absorbance by the appropriate dilution factor
and then dividing by the extinction coeffi cient of adenovirus
(
260 Ad5 = 9.09 × 10
−13
OD mL cm virion
−1
), calculated from
the data of Maizel et al. [
12
].
ε
4
Notes
1. This digestion should be as complete as possible in order to
remove the background from undigested plasmids in the fol-
lowing steps.
2. The enzymes of choice must not cut within the expression cas-
sette. As the aim of this step is to facilitate recombination, it is
recommended to leave the expression cassette by at least of
1 kb of DNA sequences on both sides. Additionally, it is better
if at least one enzyme cuts within the antibiotic resistance gene.
3. Use as controls (a) pKP1.4 linearized with
Swa
I, and (b) gel-
purifi ed fragment from the shuttle plasmid. It is recommended
to use highly competent bacteria since BJ5183 exhibit lower
transformation effi ciencies than conventional
E. coli
strains.
4. Two populations of colonies are expected: large and small size
colonies. Large colonies are generally background from the
shuttle plasmid, while small colonies will likely contain recombi-
nant plasmids, which are low copy number plasmids. The num-
ber of small colonies must be at least three times higher than in
control plate (digested pKP1.4 only) to continue the protocol.
If the number of small colonies is less than three times, start
again the procedure, and also use a different ratio in
step 5
.
5. Do not store the BJ5183 bacteria after overnight growth, as
unwanted recombinants might appear. Perform plasmid purifi -
cation early in the morning.
6. Agarose gel electrophoresis allows the identifi cation of colo-
nies containing the shuttle plasmids, which will be discarded.
Select only clones with high molecular weight DNA, as well as
those clones with no detectable DNA, since the yield of recom-
binant DNA is much lower than that from background or
unwanted rearrangements.
7. Common methods to transfect HEK293 cells include calcium
phosphate precipitation [
13
] or polyethylenimine (PEI)-
mediated DNA delivery [
8
] though other methods based on
