CaPO4 Transfection for Chromaffin Cells
1. 2M CaCl2 - 11.1g CaCl2 (anhydrous) + 47.3 ml H2O (or 14.7g CaCl2-2H2O)
Filter sterilize and store at 4'C
2. 150 mM Na2HPO4 -2.13g Na2HPO4 + 99.7 ml H2O (or 4.02 g Na2HPO4-7H20 + 97.8 ml Store at 4'C (pH is >9, nothing will grow In it)
3. 2x PlBS
280 mM NaC1 1.84g
40 mM PIPES 1.21g
1.5 mM Na2HPO4 1.0 ml of 150 mM Na2HPO4
Dissolve components in approximately 80 ml H2O. AdJust pH to 6.95 with 1.0 M NaOH. Adjust volume to 100 ml. If desired, filter sterilize. Store at 4'C N.B.: The above amounts assume the free acid ot PIPES. For NaPIPES use 1.30g.
4. DNA in TE of H2O 115 ug/ml (this is the optimum amount).
1. One to two h before DNA addition to the culture remove medium and add an amount of fresh, serum free medium (DMEM/F 12 or MEM) equal to 90% of the normal volume.
2. In a 15-ml conical centrifuge tube mix 7 parts DNA solution with 1 part 2 M CaCl2. The volume needed equals 5% of the normal culture volume. Mix gently.
3. Quickly pipet a volume of 2x PlBS equal to that of DNA+CaCl2 into the tube (from step 2)while holding on a vortex mixer set on medium speed. Continue to vortex for 1s after adding the solution. [Alternatively, the 2x PlBS can be quickly pipetted into tho bottom of the tube (no vortexing) followed by an equal volume of air released as small bubbles to thoroughly and quickly mix the solutions.] Allow tube to stand undisturbed for 30 min. EXAMPLE: For 1ml of the final DNA solution (For addition to 9 ml of culture medium) mix 82.5 ul CaCl2 with 437.5 DNA. then add 500 ul 2x PIBS.
4. Add the DNA-Ca-phosphate mixture dropwise onto the cultures. Swirl briefly to mix, Return cultures to CO2 Incubator.
5. After 4, remove the culture medium. Immediately add an amount of 12.5% g1ycerol in serum-free culture medium equal to one-half of the normal culture volume.
6. Wait exactly 3 min and add a normal volume of culture medium to the glycerol solution. Swirl to mix and aspirate. Wash the cultures once with medium, add medium and return to CO2 incubator.
Only a few dishes (<=6) or a single multi-well plate (=< 12 wells) should be glycerol shocked at one time. It is important that the cell monolayer should not become dry.