DEAE - Dextran
DEAE-DEXTRAN TRANSFECTION OF MONOLAYER OF CELLS
(TD and TS solutions)
(Greg Singer is in charge of this protocol).
This procedure is optimized for CMT3-Cos cells. It also works for CV-1 and QCL-3 quail cells. Using it for other cell types may require some modification. The rate of transfection of CMT3-Cos cells is usually about 20-30%. Instructions are given per 10cm plate (about a million cells).
Split the cells the day before the transfection to about 5 X 105 or 106 cells per plate. This is usually a 1 to 5 or 6 split of a confluent plate. IT IS CRITICAL THAT THE PLATES BE TRANSFECTED THE NEXT DAY. If the cells are on selective media, split without drug. Use Iscove's media supplemented with the normal serum that is used to grow the cells.
- Dilute the appropriate amount of plasmid DNA (usually 5-10ug) to 750ul with TS at ROOM TEMPERATURE.
- Make a 1 mg/ml DEAE-Dextran solution in TS. You need 750ul per plate. Help the DEAE-Dextran go into solution by dissolving it in a water bath at 370C. Filter the solution through a 0.45 um filter making sure that the DNA is dissolved well before filtering.
- Add an equal volume of DEAE-Dextran solution to the diluted plasmid solution and mix.
- Aspirate off medium from one Petri dish of cells (the cells should be 50-70% confluent).
- Rinse dish carefully 2X with 10ml ROOM TEMP TS. Do not leave the TS on the cells for more than a minute or two before aspirating off.
- Add the DNA/DEAE-Dextran mixture (1.5ml vol.) to the center of the dish without disturbing the monolayer.
- Incubate at room temperature for 15 minutes.
- Tilt the plates to redistribute the DNA solution and incubate at 37C for an additional 40-60 minutes with occasional tilting (like once every 15 minutes).
- Aspirate off the DNA/DEAE-Dextran mixture
- Add 3 ml 20% glycerol in TS per 100 cm plate and distribute by tilting carefully. Don't try this for more than 4-5 plates at a time. Leave on the cells for 70 seconds EXACTLY and then aspirate off. (You should examine at least a few dishes under a microscope. The cells should start to look like fried eggs. The length of time that can be tolerated by the cells will vary with each cell line and also with the state of the cells. Be very careful not to kill the cells at this step. ASK FOR HELP the first time you do this.)
- Wash the cell monolayer carefully 2X with room temperature TS to remove the glycerol completely from the cells.
- Add 10ml of warmed or room temperature Iscove's containing 100 uM Chloroquine phosphate and the normal amount of serum (usually 10%) to the monolayer.
- For regular Cos cells incubate 4 hrs. Some cell lines will only tolerate 3 hrs. ASK what is optimal for the cell line you are using from SOMEONE WHO KNOWS.
- Wash the monolayer 2X with room temperature TS.
- Replace media on the monolayer with Iscove's medium and normal amount of serum (usually 10%)
- Harvest the cells when appropriate (24- 72hrs post transfection depending on the nature of your experiment).
SOLUTIONS: Glycerol solution: Add 80ml of TS to a clean bottle. Add 20 ml of pure glycerol to this by pipetting up and down in the TS until all the glycerol is removed from the pipet. Filter through a 0.45 u filter and keep at room temperature.
Chloroquine solution: Make a 10 mM stock solution by dissolving 52 mg of chloroquine diphosphate in 10 ml of water. Filter through a 0.45 u filter and keep protected from light. Use 1 ml of this solution per 100 ml of medium.
TD (for 4 Liters):
- Solution A-
- 32g NaCl
- 1.52g KCl
- in 3.2 liters fresh H2O (taken directly from H2O system).
- Solution B-
- 0.4g Na2HPO4 (anhydrous)
- 12.0g Tris Base
- in 800ml fresh H2O
When both are into solution, add B to A, pH to 7.4-7.5 with HCl (several mls), and measure 500ml into tissue culture bottles. Autoclave 45 minutes.
TS: to make TS from TD add 0.5ml of a 20mg/ml MgCl2 and 20mg/ml CaCl2 solution per 100ml.