Biomedical Engineering Reference
In-Depth Information
3
Methods
3.1 Part I
1. Creation of viral library: Create a library of cDNAs in a choice
viral vector (
see
Note 3
). CDNAs (
see
Note 4
) can be created
by reverse transcription followed by PCR from mRNA or by
cloning from commercial cDNA libraries.
2. Purify and quantitate each plasmid. Mix equimolar amounts of
the plasmids in 13 random equal subgroups. This will make
transfection easier to handle. Ensure the concentration of the
DNA is suffi ciently high.
3. Seed AMPHO cells into fi ve 175 cm
2
fl asks so that they are
80 % confl uent in DME medium on the day of the experiment
(
see
Note 5
). Similarly, seed MCF10A into three 175 cm
2
fl asks
so they are 80 % confl uent when needed in Subheading
3.1.1
,
step 11
.
4. Trypsinize the AMPHO cells and determine cell numbers
using a hemocytometer. Dilute the cells and plate 39 dishes
with a diameter of 15 cm, each containing 7.5 × 10
6
cells, in a
volume of 20 mL. Incubate in normal tissue culture conditions
(37 °C, 5 % CO
2
).
5. On the day of transfection, ascertain that the cells are about
60 % confl uent before proceeding.
6. Prepare 13 transfection mixes, one from each library sub-
group, using the Effectene transfection reagent; gently mix
37.5
3.1.1 Creation of Viral
Library and Infection
of MCF10A
ʼ
g library plasmid DNA with 60
ʼ
L enhancer and
L buffer EC mixture in a 15 mL polystyrene tube (
see
Note 6
). Tap the sides gently to mix and incubate at room
temperature for 5 min.
7. Add 225
1,125
ʼ
L of Effectene directly into the DNA mixture, tap to
mix, and allow the Effectene-DNA complex to form by incu-
bating it at room temperature for 10-20 min.
8. In the meantime, replace the medium in the dishes with
15 mL of fresh DME medium and put the dishes back into the
37 °C incubator while waiting for the DNA-Effectene
complex to form.
9. Add 12 mL pre-warmed fresh medium to each DNA-Effectene
complex and gently mix by drawing the mixture up and down
twice with a 10 mL pipette. Gently layer one third of the
complex-medium dropwise onto the adherent cells in a dish
in a concentric spiral. This will ensure that the complex cov-
ers the entire surface area of the dish without having to swirl the
dish too much to distribute the complex uniformly. Continue
spreading the rest of the transfection mix over two more
plates, working until each transfection mix is spread over the
three dishes each. Incubate at 37 °C 5 % CO
2
for 16-24 h (
see
Note 7
).
ʼ
