Biomedical Engineering Reference
In-Depth Information
Table 2
PCR reaction mix per sample using the GoTaq Flexi Kit (Promega)
Primers: For (10
ʼ
M)
1
ʼ
L
Rev (10
ʼ
M)
1
ʼ
L
dNTP (10 mM)
1
ʼ
L
5× buffer
4
ʼ
L
MgCl
2
25 mM
2
ʼ
L
Taq polymerase
0.5
ʼ
L
DMSO
0.6
ʼ
L
DNA template (1 ng/mL)
0.5
ʼ
L
H
2
O
9.4
ʼ
L
ʼ
Total volume
20
L
4. Visualize and document the PCR products by UV light. Excise
each band individually, and purify the PCR fragment using
QIAEX II Gel Extraction System (Qiagen;
see
Notes 19
and
20
).
5. Sequence the individual PCR fragments with the vector-spe-
cifi c primer gcctcctcttcttccatccg. The sequencing results (
see
Note 21
) are subjected to nucleotide BLAST analysis on the
NCBI web site:
http://blast.ncbi.nlm.nih.gov/Blast.cgi
[
12
]
for insert identifi cation.
3.2 Part II:
Reconstructive
Analysis by Direct
Viral Infection
Viral supernatant carrying selected recombinant genes is produced
and used in combinations to infect MCF10A cells for verifi cation.
Integration of the genes into the genome is confi rmed by direct
sequencing or their expression by immunoblotting.
1. From 75 cm
2
fl asks of 80 % confl uent AMPHO cells in DME
medium, trypsinize the cells, resuspend in fresh medium, and
count with a hemocytometer and seed at 2.5 × 10
6
cells per
10 cm diameter dish. Allow to grow overnight at 37 °C, 5 %
CO
2.
The confl uency of cells in each dish should be about 60 %
as viewed by microscopy on the day of transfection.
2. Add 5
3.2.1 Direct Infection
and Stable Recombinant
MCF10A Cells
ʼ
g of DNA of the plasmid construct to a 16
ʼ
L enhancer
L buffer EC mixture in a 5 mL polystyrene (PS) tube
(
see
Note 6
). Tap the sides gently to mix, and incubate for
5 min at room temperature.
3. Add 60
and 300
ʼ
L of Effectene directly into the DNA mixture, tap to
mix, and allow the Effectene-DNA complex to form by incu-
bating it for 10-20 min.
ʼ
