| Contributor: |
The Laboratory of Daniel Portnoy at the University of California, Berkeley
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1. Use COS cells that are approximately 75% confluent.
2. Dilute DEAE-dextran in COS Media 1 to 250 μg/ml.
3. Dilute DNA to be transfected into cells to 4 μg/ml in DEAE-dextran/COS 1 solution (see Hint #2). Mix well.
4. Aspire the media from COS cell plates and wash twice with pre-warmed (37°C) PBS.
5. Aspirate the PBS from the COS cell plates and wash once with pre-warmed COS Media 1.
6. Remove the COS Media 1 from the plates.
7. Add 3 ml of DEAE-dextran/DNA/COS Media 1 (made in Step #3) to each of the COS cell plates to be transfected.
8. Incubate for 90 min at 37°C, shaking the plates every 15 minutes (see Hint #3).
9. Remove the DEAE-dextran/DNA/COS media 1 (see Hint #4).
10. Treat each plate with 5 ml of DMSO-COS 3 media for 3 min (see Hint #5).
11. Aspirate the DMSO-COS 3 media and wash the plates twice with pre-warmed COS Media 1.
12. Incubate each plate at 37°C with 5 ml of Chloroquine-COS 3 Solution for 5 to 8 hours (see hint #6).
13. Aspirate the Chloroquine-COS 3 Solution and wash the plates twice with pre-warmed COS Media 1.
14. Add 10 ml of COS 3 Media per plate.
15. Harvest transiently transfected cells 36 to 40 hours later and assay for expression of the transfected DNA.
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| Chloroquine (100X) |
| Sterilize.
Store at -20(C protected from light
10 mM Chloroquine
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| DMSO-COS 3 |
| 10% (v/v) DMSO (CAUTION! see Hint #1)
Prepare just before use
Prepared in COS Media 3
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| PBS |
| Sterilize
pH 7.2
1.8 mM Potassium Phosphate Monobasic (K2HPO4)
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
137 mM NaCl
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| L-Glutamine (1X) |
| 30 mg/ml L-Glutamine
Sterile
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| COS Media 3 |
| 100 μg/ml Streptomycin Sulfate
100 Units/ml Penicillin G
DMEM
1X L-Glutamine
10% (v/v) Fetal Calf Serum
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| COS Media 1 |
| 100 μg/ml Streptomycin Sulfate
100 Units/ml Penicillin G
1X L-Glutamine
Dulbecco's Modified Eagle Medium (DMEM)
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| DEAE-dextran |
| 50 mg/ml DEAE-dextran
Autoclave to sterilize.
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| Chloroquine-COS 3 Solution |
| 100 μl of 100X Chloroquine
10 ml COS Media 3
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Potassium Phosphate, Monobasic
Fetal Calf Serum (FCS)
DMEM
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Chloroquine
DEAE-dextran
Penicillin G
Streptomycin Sulfate
L-Glutamine
DMSO
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. The amount of DNA needed to achieve maximal transient expression will need to be determined empirically. Large amounts of DNA can be inhibitory.
3. Shaking the plates every 15 min is a key to the transformation success. Check the cells under a microscope periodically. Stop the incubation when the cells appear rounded and to have shrunk.
4. Do not wash the plate so that you do not remove the DNA.
5. Treat only 2 plates at a time so that incubation times are accurate. DMSO is extremely toxic to cells.
6. The optimum time is 7 hours.
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