APPENDIX C
Transient transfection
Calcium phosphate (CaPi) method
Reagents
1. 2.5M CaCl2
Composition per 40 ml
CaCl2 (MW 111g)
11.104 g
MQ
40 ml
2. 2X HBS
Composition per 250 ml
NaCl
4g
KCl
0.185 g
Na2HPO4
53.25 mg
Dextrose
0.5 g
HEPES
3.25 g
DPBS
225 ml
Adjust pH with NaOH to 7.05
Adjust volume to
3. 0.1X TE buffer
Composition per 40 ml
1X TE buffer
DPBS
40 µl
40 ml
250 ml
Protocol
For 6-well plate, HEK293T cells
Day 0
1. Seed cells into container and incubate until get 50-60%
confluent.
Day 1
2. Re-feed the cells with fresh complete medium contained
100ng/ml Doxycycline
(per 2 ml medium: 1,940 µl medium + 60 µl of 5 µg/ml
Dox)
3. Prepare CaPi-plasmid solution per well, respectively.
Plasmid
1 µg
2.5 CaCl2
0.1X TE buffer
15 µl
add up to 150 µl
4. Bubbling the CaPi-plasmid solution while adding 150 µl of
warm 2X HBS and gently vortex.
5. Incubate the mixture 20 minute at 37°C
6. Add the mixture 300 µl/well
7. Incubate for 16-20 hr.
Day 2
8. Re-feed the cells with fresh complete medium contained
100 ng/ml Dox and 3 µg/ml Puro
(per 2 ml medium: 1,880 µl medium + 60 µl 5 µg/ml Dox +
60 µl 100 µg/ml Puro)
Day 3 or Day 4
9. After 48 or 72 hr., harvest the transfected cells for analysis
(both for mRNA and Protein)
**** HEK293T cells have high transfect efficiency reach up
99%, there are not necessary for Puromycin selection)
Polyethylenimine (PEI) method
Reagents
1. 0.5 mg/ml PEI
Composition per 50 ml
PEI
2.5 g
MQ
50 ml
2. 150mM NaCl
NaCl
MQ
7.305 g
25 ml
Dilute with 450 µl of 5 M NaCl with DPBS to 15 ml total
volume. Filtered through 0.2 µm filter then aliquot into 15 ml
tube and stored at 4°C.
Protocol
For 6-well plate, HEK293T cells
Day 0
1. Seed cells into container and incubate until get 50-60%
confluent.
Day 1
10.
Re-feed
the
cells
with
fresh
complete
medium
contained 100ng/ml Doxycycline
(per 2 ml medium: 1,940 µl medium + 60 µl of 5 µg/ml
Dox)
2. Prepare PEI-plasmid solution per well
Plasmid
1 µg
0.5 mg/ml PEI
4 µl (1:4 w plasmid/v)
150 mM NaCl
add up to 150 µl
3. Incubate the mixture 20 minute at room temperature
4. Add the mixture 150 µl/well
5. Incubate for 16-20 hr.
Day 2
6. Re-feed the cells with fresh complete medium contained
100 ng/ml Dox and 3 µg/ml Puro
(per 2 ml medium: 1,880 µl medium + 60 µl 5 µg/ml Dox +
60 µl 100 µg/ml Puro)
Day 3 or Day 4
7. After 48 or 72 hr., harvest the transfected cells for analysis
(both for mRNA and Protein)
Chemical reagents for transfected cells
1. 1 mg/ml Doxycycline
Doxycycline
MQ
0.005 g
5 ml
Filtered through 0.2 µm filter then aliquot into 1.5 ml
tube and stored at
-30°C.
2. 1 mg/ml Puromycin
Puromycin
MQ
0.005 g
1 mg/ml
Filtered through 0.2 µm filter then aliquot into 1.5 ml
tube and stored at
-30°C.