Download CaPhos. Transfection Protocol

CaPhosphate - DNA Precipitate Transfection Protocol
for CHO and L6 cells
Solutions:
2.5 M CaCl2
183.7 g CaCl2 dihydrate
H2O to 500 ml
filter sterilize, aliquot, freeze.
2 X Hepes Buffer Saline Soln
16.4 g NaCl
11.9 g Hepes Acid
0.21 g Na2HPO4
800 ml H2O
titrate to pH 7.05 with NaOH
add H2O to 1 L
filter sterilize, aliquot, freeze.
1.
Split cells 1:15 the day before the transfection. Feed cells with complete
media 2-4 hours before the precipitation.
2.
EtOH precipitate 10-50 µg of DNA and air-dry the pellet in hood.
Resuspend pellet in 450 µl sterile DDH2O. Add 50 µl of 2.5 M CaCl2
(sterile).
3.
Place 500 µl of 2 X HeBS in a sterile 15 ml falcon tube. Use a pipettor
attached to a plugged 1 ml pipet to bubble the 2 X HeBS and add the
DNA/CaCl2 solution dropwise with a pasteur pipet. Immediately vortex
for 5 seconds.
4.
Allow precipitate to sit 20 min at RT.
5.
Use pasteur pipet to distribute the precipitate evenly over a 10 cm dish
of cells and gently swirl to mix.
6.
Incubate the cells 4-16 hours. Remove the media. Wash cells 2 X with
1 X PBS. Put on 10% DMSO in PBS to shock for 3 minutes at RT. Add
PBS to dilute and aspirate. Wash cells 2 X with 1 X PBS. Put on fresh
media.
7.
For transient transfection, harvest cells 2 days after. For stable
transfection, allow cells to double 2 X before adding selective media.