Biomedical Engineering Reference
In-Depth Information
7 Transfer the mixture into pre-warmed complete media (210 ml) drop by drop.
8 Dispense 22ml of media from step 7 into each 15 cm dish immediately.
9 Equalize by forth and back motion.
10 Incubate at 37
◦
Cand5%CO
2
in an incubator for about 48 h.
Notes
a
Using low passage number (
<
P50) of HEK 293 cells.
b
An exact pH of 2
×
HBS is extremely important for efficient transfection. The optimal pH range
is 7.05-7.12.
c
About 70-80% of confluency is ideal.
d
Discard old media before adding 2
×
HBS.
e
This is the key step for transfection. Check Ca3(PO4)2 precipitate when you prepare new 2.5
M
CaCl2 and 2
×
HBS.
PROTOCOL 12.2 Harvesting Transfected Cells
for 10 of 15 cm Dishes
Equipment and reagents
•
Cell scraper
•
DMEM (Cellgro)
•
FBS
•
Antibiotic-antimycotic (100
×
) (Invitrogen)
•
50ml conical tubes
•
Pipettes (5, 10 and 25 ml)
•
Centrifuge
•
Biological safety cabinet
•
Lysis buffer: 150m
M
NaCl and 50m
M
Tris (pH 8.4).
Method
1 Take the dishes from the CO
2
incubator and scrape them with cell scrapers to dislodge
all cells with media.
2 Collect cells and media in six 50ml conical tubes.
3 Add 30ml of fresh media to the dishes (one by one) to rinse remaining calls and
distribute the media into the above six 50 ml conical tubes.
4 Centrifuge the conical tubes at 1000 rpm, 4
◦
C for 10-15min to pellet the cells.
5 Discard supernatant.
