DEAE DEXTRAN TRANSFECTION METHOD
Solutions:
10 mg/ml DEAE Dextran
2 X 106 MW Dextran
Make up in TBS
filter sterilize and aliquot
store at 4 C
Tris-buffered Saline (TBS)
Soln A
Soln B
80 g/L NaCl
15 g/L CaCl2
3.8 g/L KCl
10g/L MgCl2
2 g/L Na2HPO4
filter-steriliz
30 g/L Tris base
store at -20 C
Adjust pH to 7.5
Filter sterilize
store at -20 C
For 100ml, add 10 ml solution A to 89
ml H2O. While stirring rapidly, add 1
ml solution B slowly, drop by drop.
Filter sterilize and store at 4 C.
1.
Plate cells on coverslips in 6 well dishes at 1.92 X 105 cells/well. Grow
overnight.
2.
Prepare DNA in 2 ml total volume. 0.5 µg DNA/well
[DEAE Dex]
400 µg/ml
200
100
50
DNA vol in TBS
40 µl
20 µl
10 µl
5 µl
DEAE Dex vol.
80 µl
40 µl
20 µl
10 µl
Total
120 µl
60 µl
30 µl
15 µl
You will need to determine which concentration of DEAE Dextran is
optimum for your cell type. Always do 2 wells of each DNA, and
always do a blank control, as well as a positive control. Make sure the
DNA is sterile as well. This can be done by EtOH precipitating the DNA
and washing with 70% EtOH, and then let the pellet dry in the
hood.
Resuspend in sterile TE.
3.
Aspirate the media off wells, wash with 2 ml of 1 X PBS. Add 2 ml of
DMEM + 100 µM chloroquine. This facilitates the cells uptake of the
foreign DNA.
4.
Add the DNA - DEAE Dextran mixture dropwise to the well. Swirl
thoroughly.
5.
Incubate wells 4 hours at 37 C.
6.
Aspirate media
7.
Shock cells with 2 ml of 10% DMSO in 1 X PBS. Incubate for 1 minute
at RT. Add 10 ml of 1 X PBS to dilute, then aspirate. Wash well with 3
ml of 1 X PBS. Add 2 ml DMEM to well.
8.
Grow several days, fix coverslips and immunostain.
DEAE Dextran Concentration for several different cell types:
L Cells
Cos Cells
200 µg DEAE Dextran concentration
400 µg DEAE Dextran concentration