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MOLECULAR BIOLOGY: WORKING WITH DNA

INTRODUCTION OF DNA INTO CELLS

CALCIUM PHOSPHATE TRANSFORMATION OF MAMMALIAN CELLS

Calcium Phosphate Transformation of Mammalian Cells Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of J. Michael Bishop at the University of California, San Francisco
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
This protocol describes a technique for introducing plasmid DNA into tissue culture cells. The uptake of DNA by mammalian cells is facilitated by the formation of a calcium phosphate-DNA coprecipitate. The use of BES Buffer produces more reliable results than HBS. BES at pH 6.95 and a CO2 level of 3% has been reported to be optimal.
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. For each 100 mm plate of cells to be transformed, precipitate 20 μg DNA with Ethanol and pellet by centrifugation. 2. Rinse the DNA pellet once with cold 80% Ethanol and pellet the DNA by centrifugation. 3. Decant the Ethanol and evaporate it from the pellet by air-drying it in a laminar flow hood for about 15 min. 4. Dissolve the DNA in 450 μl of sterile TE Buffer (see Hint #2). Add 50 μl of 2.5 M CaCl2 and mix. 5. Slowly add the 500 μl of DNA solution to 500 μl of 2X BES-buffered saline (see Hint #3). Incubate at room temperature for 20 min to allow the Calcium Phosphate-DNA complex to form. 6. Add the Calcium Phosphate-DNA mixture drop-wise to cultured cells and incubate at 37°C (see Hint #4). 7. Aspirate the medium and add 37°C TBS with 25% DMSO (see Hint #5). Incubate for 30 sec. 8. Quickly aspirate and gently rinse the plates twice with PBS, and feed the cells with fresh medium. 9. For drug selection, split the cells one or two days after DMSO shock.
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
2.5 M CaCl2    Filter sterilize and store at -20°C 80% (v/v) Ethanol PBS    pH 7.2 4.3 mM Na2HPO4 2.7 mM KCl 1.8 mM Potassium Phosphate Monobasic (KH2PO4) 137 mM NaCl TBS with 25% DMSO    150 mM NaCl 100 mM Tris, pH 7.5 25% (v/v) DMSO (CAUTION! See Hint #1) TE Buffer    10 mM Tris pH 8.0 1 mM EDTA BES-Buffered Saline (2X)    280 mM NaCl Filter sterilize and store at -20°C 1.5 mM Sodium Phosphate Dibasic (Na2HPO4) 50 mM BES, pH 6.95
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
BES Potassium Phosphate, Monobasic Calcium Chloride Tris Ethanol Sodium Phosphate, Dibasic Potassium Chloride Sodium Chloride EDTA DMSO
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. If desired, CsCl-purify the DNA and treat with RNase. 3. While adding the DNA, vigorously agitate the solution with a stream of air though a plugged, sterile Pasteur pipette. 4. For highest transfection efficiency, use exponentially-growing cultures (at least four days from thawing cells or splitting from high density). Incubation time may have to be determined empirically for specific cell lines. A very fine precipitate should be observed on top of the cells within 1 hr after the addition of the Calcium phosphate-DNA complex. 5. The DMSO shock may increase transfection efficiency from approximately 0.1% up to 1 to 2%. However, cell mortality as high as 50% may occur.
Citation and/or Web Resources Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell Biol. 1987; 8:2745-52.